Immunohistochemistry

(IHC) Handbook
Data image on handbook cover provided by Ashley Oliver (Senior Research
Associate, Antibody Development)

Detection of TGF beta mRNA (white), Arginase 1 protein (red), and CD204/
SR-AI/MSR protein (green) in human prostate cancer tissue using Integrated
Co-Detection Workflow.
Introduction to Immunohistochemistry 2
Multiplexing 4

ISH-IHC (RNA-Protein Co-Detection) 4

IHC Workflow 8
Sample Preparation, Fixation, and Sectioning 8
Tissue Processing: Formalin-Fixed, Paraffin-Embedded (FFPE) vs. Frozen 8
Fixation 9
Protocol: Formalin-Fixed, Paraffin-Embedded (FFPE) 10
Protocol: Method I - Frozen Samples (Whole Animal or Dissected Tissue) 11
Protocol: Method II – Cryopreservation & Sectioning of Alcohol-Fixed Tissues 12
(Epitope) Antigen Retrieval 14
What is Antigen Retrieval? 14
Is Antigen Retrieval Always Necessary? 15
Protocol: Heat-Induced Epitope Retrieval (HIER) 15
Protocol: Proteolytic-Induced Epitope Retrieval (PIER) 15
Blocking Non-Specific Binding 17
Endogenous Activity and Reactive Epitopes 19
Antibodies and Detection Methods 21
Antibody Clonality: Polyclonal vs. Monoclonal 21
Detection Methods 23
IHC Staining: IF and Chromogenic 25
Protocol: Deparaffinization & Rehydration for Paraffin-Embedded Tissues 25
Protocol: IHC Staining 25
Protocol: IF Staining 26
Protocol: Chromogenic Staining 27
IHC Controls 27

Visual Workflow 30
Traditional IHC 30
ISH-IHC (RNA-Protein Co-Detection) 32

Troubleshooting Guide 32

Basic Buffer & Reagent Recipes 36

Sample Preparation & Fixation 36
Antigen Retrieval Buffers 36

References 37
Additional Resources 37

Immunohistochemistry Handbook - Fall 2022

This handbook is intended to serve as a starting point for understanding the principles behind
Immunohistochemistry (IHC). Use this guide as a reference for understanding, performing, and
troubleshooting IHC protocols, while developing and optimizing IHC assays.
Introduction to
Immunohistochemistry
Immunohistochemistry (IHC) and immunocytochemistry The most challenging aspect of IHC is determining and
(ICC) are techniques that use antibodies to detect antigens optimizing the experimental conditions required to generate
and provide semi-quantitative data about target protein a strong and specific signal for each antigen of interest. While
expression, distribution, and localization. Both IHC and the technique is relatively straightforward in principle, there
ICC are dependent on specific epitope-antibody interactions, are many variables to consider in a given experiment.
but IHC refers to the use of tissue sections whereas ICC
is performed on cultured cells or a cell suspension. The For example, visualization of an abundant protein in
principles of IHC date to the 1930s and were first reported formalin-fixed tissue will likely require antigen retrieval
in the literature in 1942 when Dr. Albert Coons used a and may be compatible with direct detection using a
fluorescein-labeled anti-pneumococcal antibody to fluorochrome-conjugated primary antibody. In contrast,
identify bacteria. detection of a phosphorylation-dependent epitope in a
section of frozen tissue may require signal amplification
In both of these methods, positive marker staining is and additional blocking steps.
visualized via a molecular label, which can be chromogenic
(enzymatic) or fluorescent. Briefly, samples are fixed to
preserve cellular integrity and then subjected to incubation
with blocking reagents to prevent non-specific binding of
the antibodies. Samples are subsequently incubated with
primary and often secondary antibodies, and the signal is
visualized with a microscope

Figure 1. Traditional Chromogenic IHC Workflow. Enzymatic conversion of a chromogen substrate is required to visualize an antigen in traditional IHC assays. As
an alternative, fluorescence detection can be used and is more suitable for multicolor experiments given the range of available fluorochromes and emergence of
high contrast imaging.

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Variables Influencing Experimental Design and Optimization

Variable Factors

Antigen/Target Species, expression levels, sample types, and subcellular location

Tissue Species, type, section size, and thickness

Epitope Dependence on conformation or post-translation modification

Sample Preparation Paraffin-embedded or frozen

Fixation Method Perfusion or immersion (with or without freezing)

Fixative Aldehydes (crosslinking), alcohols (precipitating), or acetone

Normal serum, BSA, casein, various detergents,

Blocking Reagent
and salt concentrations

None, Heat-induced Epitope Retrieval (HIER),

Antigen Retrieval
and Protease-induced Epitope Retrieval (PIER)

Permeabilization Triton-X 100, Tween-20, or saponin

Detection Method Direct or indirect (with or without signal amplification)

No primary antibody, isotype control, absorption control,

Appropriate Controls
tissue type control

Primary Antibody Monoclonal or Polyclonal

Secondary Antibody Species reactivity and label

Simultaneous or sequential antibody addition,

Multiplex
and antibody host species

Labeling Method Fluorescence or chromogenic

Fluorochromes (numerous options)

Label Chromogenic reagents: enzymes (HRP, AP) and substrates
(DAB, AEC, NBT/BCIP)

Fluorescence: DAPI, Hoechst 33342

Counterstain
Chromogenic: Hematoxylin, Fast Green FCF

Fluorescence: anti-fade mounting medium

Mounting Reagent
Chromogenic: aqueous mounting medium

Visualization and Analysis Fluorescence microscope or standard light microscope

The above table is not intended to be exhaustive but rather summarizes common factors influencing IHC experimental design at Bio-Techne.

3
Multiplexing
The emergence of multiplex IHC has enabled a more While IHC multiplexing allows for detection of multiple
comprehensive analysis of protein expression, complex target proteins at the same time, it can also be combined
cell-cell interactions, and spatial distribution of cell with in situ hybridization (ISH) for detection of both RNA
populations. While flow cytometry analysis also allows and protein within the same tissue. ISH-IHC analysis
for detection of multiple markers within a cell population, provides sensitive detection, characterization, and
it lacks contextual information on the interaction between localization of nucleic acids and proteins while conserving
markers or their spatial relationship. Multiplexing IHC the cell and tissue structural integrity.
is particularly advantageous when studying the tumor
microenvironment (TME) for its prognostic and diagnostic
benefits. The ability to spatially resolve multiple biomarkers
simultaneously improves the understanding of the complex
microenvironment consisting of various cell types, cytokines
and growth factors, inflammatory factors, and extracellular
matrix components. Additionally, regarding the TME,
multiplexing can improve clinicians’ ability to stratify cancer
patients and better predict patient outcomes.

Multiplexed antibody-based imaging methods are

categorized based on the type of antibody tag, such as
fluorophores, metal ions, and DNA oligonucleotide barcodes.
This technique can be performed using either sequential or
simultaneous staining methods. Multiplex cycling platforms
such as the Akoya Biosciences® PhenoCycler®, formerly
CODEX®, which relies on DNA barcodes, and Canopy
Biosciences® ChipCytometry, which uses fluorescently
conjugated antibodies, employ consecutive cycles of reporter
addition/removal or staining/bleaching, respectively.
Imaging Mass Cytometry™ (IMC; Fluidigm) and multiplex
ion beam imaging (MIBI) are two common technologies that Figure 2. IMC multiplexed antibody image in human tonsil. Detection of
utilize metal-conjugated antibodies and allow simultaneous markers using metal-conjugated antibodies in human tonsil: CD19-142 Nd
(magenta), CD38-141 Pr (lime), Collagen Type I-169 Tm (yellow),
imaging of over 40 biomarkers in a tissue section at E-Cadherin-158 Gd (cyan; R&D Systems, Catalog # AF748),
single-cell resolution. Ki670168 Er (red). Image courtesy of verified customer review.

ISH-IHC
(RNA-Protein Co-Detection)
ISH is a technique that employs probes to detect specific The proprietary double Z probe design amplifies target-
DNA or RNA sequences in heterogeneous cell populations specific signals while suppressing background noise from
such as fixed tissue sections or cells. RNA ISH reveals spatial non-specific hybridization. RNAscope ISH technology can be
information about gene expression within the sample. This employed across a number of research areas such as cancer,
technique is advantageous when there is no ideal antibody immuno-oncology, neuroscience, immunology, and more.
for a protein or protein expression is too low and an antibody Common targets for ISH include cytokines and chemokines,
is not sensitive enough. Additionally, ISH is particularly tumor markers, neuronal genes, splice variants, immune cell
useful for identifying the cellular origin of secreted proteins, activation markers, and viral RNA.
like cytokines and growth factors. ACD’s RNAscope™
technology is a highly sensitive and specific ISH assay for
single molecule RNA detection with single-cell resolution.
 RNAscope miRNAscope BaseScope DNAscope

mRNA Small non-coding RNAs Exon junctions, splice Gene copy number
including miRNAs, ASOs, variants, short/highly variations (amplifica-
Molecule Type and siRNAs homologous RNA tions/deletions) and
sequences, and point gene rearrangements/
mutations fusions

>300 nt 17-50 nt 50-300 nt >3000 nt (Vector/Viral)

and
Target Size
>20,000 nt
(Chromosomal)

Image
Example
RNAscope™ miRNAscope™ assay Splice Variant Example DNAscope chromogenic
HiPlex v2 12-plex used to specifically showing detection duplex (red/blue)
target-specific marker detect miR-138-5p of EGFRvIII+ in staining in ALK
probes used to detect in Purkinje cells in glioblastoma with Break-apart positive
immune cells, tumor cerebellum of the BaseScope v2 assay. cell line using the target
cells, chemokines and mouse brain. probes HS-ALK-BA-5’/
cytokines in ovarian HS-AK-BA-3’. Break apart
cancer tissue. events are detected
through appearance of
blue dots.

Figure 3. In situ detection of CD4+FoxP3+ Regulatory T cells (Tregs) in human breast cancer using RNAscope
RNA-protein co-detection assay. CD4 mRNA (red) and FOXP3 protein (green) were detected in FFPE tissue sections of
human breast cancer. ACD's Integrated Co-Detection Workflow was performed using ACD RNAscope Probe Hs-CD4 (ACD
Catalog # 605608) and Rabbit Anti-FoxP3 Polyclonal Antibody (Novus Biologicals Catalog # NB600-245) at 1:200 dilution.
Tissue was stained on Leica Bond RX using RNAscope™ 2.5 LS Reagent Kit-RED (ACD Catalog # 322150), BOND Polymer
Refine Detection (DAB) and Hematoxylin, BOND Polymer Refine Red Detection and Hematoxylin and RNAscope™ 2.5 LS
Green Accessory Pack (ACD Catalog # 322550). Tissue was counterstained with 50% hematoxylin (blue).

When used alongside IHC, ISH provides a more comprehensive overview of the complex molecular mechanisms involved in
biological pathways. ISH and IHC techniques share a similar workflow including sample fixation and signal amplification. Thus,
methods have been developed to collect RNA and protein expression data on the same tissue sample, referred to as ISH-IHC or
RNA-protein co-detection, which can be performed either sequentially or following an integrated co-detection protocol.

5
 Figure 4. Detection of Arginase 1 protein (red), CD204/SR-AI/MSR
Applications and Benefits of ISH-IHC protein (green) and TGFβ1 mRNA (white) in human prostate cancer tissue
using Integrated Co-Detection Workflow.
• Validate Antibody Specificity – RNA-protein ACD’s Integrated Co-Detection Workflow (ICW) was performed on
co-detection can be used to validate the specificity formalin-fixed paraffin-embedded tissue sections of human prostate
of the antibody by simultaneously comparing the cancer using Rabbit Anti-Human Arginase 1 Polyclonal Antibody
(Novus Biologicals, Catalog # NBP1-32731) at a 1:50 dilution, Goat
localization of the RNA and protein signal for the Anti-Human CD204/SR-AI/MSR Polyclonal Antibody (R&D Systems,
same target, which qualifies as orthogonal Catalog # AF2708) at 3 μg/mL, and ACD RNAscope Probe Hs-TGFβ1
(Advanced Cell Diagnostics, Catalog # 400888). Tissue was stained
antibody validation. using Donkey Anti-Rabbit IgG NorthernLights™ NL557-conjugated
Antibody (R&D Systems, Catalog # NL004), Donkey Anti-Goat IgG
• Visualize Source of Secreted Proteins – Target NorthernLights™ NL493-conjugated Antibody (R&D Systems,
Catalog # NL003), RNAscope™ LS Multiplex Fluorescent Reagent Kit
the transcript of secreted proteins, such as cytokines (Advanced Cell Diagnostics, Catalog # 322800), and TSA Cyanine 5
or chemokines, and use protein markers to identify (Akoya Biosciences, Catalog # NEL745001KT). Tissue was
counterstained with DAPI (blue).
the cellular source.

• Ascertain Marker Expression, Activation,

and Spatial Mapping – Detection in a
Learn More About RNAscope™
morphologically relevant context is used to assess Technology From Bio-Techne
cell-cell interactions (i.e. hormone-receptor
interaction, endocytosis or excretion). Immune cells
that infiltrate the tissue microenvironment (TME)
can be characterized using activation markers
detected by ISH and immune cell markers such as
CD3, CD4, CD8, CD45, and CD68 by IHC. Similarly,
this approach can be applied to brain mapping to
detect gene expression in neuronal subtypes using ISH
alongside standard neuronal markers by IHC.

• Increased Multiplexing Capability - Several

markers are visualized simultaneously in tissue and
RNAscope probes for ISH can be employed when an
adequate antibody is not available, or protein
expression is too low to detect by traditional IHC.

• Identify Transcript Variants – Detect cell-type

specific expression of splice variants and
gene mutations.

• Visualize small RNAs – Study expression of microRNAs

(miRNAs) implicated in cancer or track small interfering
RNA (siRNA)/antisense oligonucleotides (ASO)
therapeutics in pre-clinical models by combining
cell-marker specific antibodies.
IHC
WORKFLOW
Sample
Preparation, Fixation,
and Sectioning

7
Sample Preparation, Fixation,
and Sectioning

Tissue Processing: Formalin-Fixed,

Paraffin-Embedded (FFPE) vs. Frozen

IHC can be broadly classified into two forms based on the While paraffin embedding is thought to better preserve
type of tissue processing involved: IHC- formalin-fixed, morphological details and offers the best option for
paraffin-embedded (FFPE), and IHC-frozen (Fr). Often long-term preservation of tissue samples, cryopreservation
the preservation method is closely associated with the is considered to better preserve enzyme and antigen
type of fixation. Formalin-fixed tissues are commonly expression. The optimal method for each experiment should
paraffin-embedded following fixation, while frozen tissue be determined by considering the nature of the antigen, its
sections can be fixed with formaldehyde or alcohol prior to subcellular location, and desired method of detection, among
or following cryosectioning. other factors.

Formalin-Fixed, Paraffin Embedded (FFPE) Frozen

Fixation Prior to embedding into paraffin Performed either before or after cryosectioning

Common Fixative Formaldehyde Formaldehyde or Alcohols

Sectioning Microtome, 4-10 µm sections Cryostat/Cryotome, 5-20 µm sections

Ideally, fresh sections should be cut Short-term: 1 year at -80°C

after 4 weeks due to loss of antigenic
Storage epitopes. For long-term storage (several
years), coating of slide in paraffin
is recommended.

· Ease of handling · Preserves enzyme and antigen function

Advantages · Preserves structural morphology · Useful for study of post-translationally
· Blocks can be stored long-term modified protein, DNA, or RNA

· Variable fixation times · Less optimal for studying structural morphology

· Fixation can mask epitopes · Ice crystals may impact tissue structure
Limitations · Endogenous enzymatic activity may impact the
IHC detection method

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Paraffin-Embedding Tissue Fixation
Because paraffin is immiscible with water, tissue must be
dehydrated before adding molten paraffin wax. Dehydration All samples used in an IHC experiment must be fixed.
is achieved by immersion in increasing concentrations The fixation process preserves the histologically relevant
of alcohol. This approach allows for a gradual change in morphology of tissues for future analysis and maintains
hydrophobicity and minimizes cell damage. Following antigenicity of target molecules by preventing autolysis
dehydration, the tissue is incubated with xylene to clear any and target protein degradation. Fixation alters the chemical
remaining ethanol. Paraffin is typically heated to 60 °C for composition of tissues and often requires a compromise
embedding and is then allowed to harden overnight. between preserving tissue structure and preserving the
epitope. For the best results, tissue should undergo rapid and
The tissue is subsequently cut with a sharp blade into uniform fixation.
ultra-thin slices using a microtome. Sections are then
Whole animal perfusion (Figure 5A), using an animal’s
dried onto microscope slides and can be stored at room
vascular network to disperse the fixative solution, is the
temperature for extended periods of time. Tissue sections
preferred method for tissues from small animals including
must be rehydrated before commencing the IHC protocol.
mice and rats. Alternatively, dissected tissue or organs can
Paraffin is the cheapest and most commonly used substance
be directly immersed in the fixative immediately after tissue
for tissue embedding. However, tissue can also be embedded
collection. (Figure 5B).
in plastic, which sets harder and allows sectioning of thinner
tissue slices (1.5 µm versus 5 µm).

Figure 5. Fixation Methods. Perfusion (A) versus Immersion (B)

Fixation Method Specifications When To Use It Advantages Disadvantages

Perfusion of 4% For large, intact tissue · Rapid dispersion of · Technically challenging

paraformaldehyde of small animals. fixative throughout · Time consuming
solution through the the animal
Perfusion Fixation vascular system. · Uniform fixation of
large organs

Immersion of dissected For small pieces · Quick · May result in

tissue (~10 mm thick) of dissected tissue. · Technically simple incomplete fixation
at room temperature of large tissue
Immersion Fixation in fixative volume · May fail to penetrate
50-100x greater than all regions of tissue at
the tissue volume.
the same rate

9
 Type of Fixatives

Aldehydes 4% formaldehyde in phosphate-buffered saline (PBS) is the most common fixative for preserving protein targets
in tissues. Formaldehyde reacts with amino groups in proteins to form methylene bridges that crosslink proteins
in tissue sections. These molecular crosslinks can mask protein epitopes from antibody binding and may require
the addition of an antigen retrieval step to allow antibody access to the epitope. Additionally,
formaldehyde-mediated tissue fixation has been shown to induce translocation of phosphorylation-dependent
epitopes from the membrane to the cytoplasm.

Alcohols The predominant alcohols used for fixation are ≥70% methanol and ≥80% ethanol. Alcohols work by removing
and replacing water molecules in tissue, which can destabilize hydrophobic bonds and alter the tertiary structure
of proteins. This also causes the precipitation of soluble proteins, making alcohol-mediated fixation more
appropriate for detection of membrane bound proteins. Alcohol, unlike formaldehyde, does not mask epitopes,
thus antigen retrieval does not need to be done on tissue fixed with alcohol.

Acetone
Acetone is also used as a strong dehydrant and precipitant, typically applied to sections of snap-frozen tissues.
Acetone fixation is generally mild and may be followed by fixation with alcohols or formaldehyde.

No single fixation condition works for all target molecules

and tissues. The duration and method of fixation can impact
preservation, tissue integrity, and antibody binding capacity. What’s the difference between
paraformaldehyde, formaldehyde, and formalin?
Ideal fixation conditions should be determined to optimally
Paraformaldehyde (PFA) is the polymerized form of
preserve tissue for each experiment. Incomplete fixation
formaldehyde and is not itself a fixing agent. Formaldehyde
(under-fixation) of cells or tissues may allow rapid proteolytic
can be prepared by dissolving PFA in PBS using heat and
degradation of target proteins within the tissue and can
sodium hydroxide (NaOH). Formalin refers to a saturated
reduce specific immunoreactivity. Any issues arising from
formaldehyde solution and some commercial formalin
incomplete fixation, such as autolysis, cannot be reversed or
solutions include methanol as a stabilizer to prevent
fully rectified in later steps. Excessive fixation (over-fixation)
formaldehyde polymerization. A 10% formalin solution is
may result in masking of the epitope or in strong non-specific
equivalent to a 3.7% formaldehyde solution.
background staining that can obscure specific labeling.

Protocol: Formalin-Fixed, Parrafin-Embedded (FFPE)

1. Fix the tissue of interest by immersing it in 10% neutral buffered formalin (4% formaldehyde) for 4-24 hours at room
temperature. Fixation time and temperature depends on tissue type/size. After fixation, wash the tissues 3x in PBS.

Note: It is not recommended to fix tissue for >24 hrs because over-fixation can cause masking of the
antigen. If necessary, tissues can be transferred to alcohol after fixation prior to starting the
embedding process.

Note: Formaldehyde-based solutions should be aliquoted and frozen, or stored at 4–8°C for up to one month.

2. Dehydrate by full immersion of tissue in the following solutions (2x for 30 minutes each):
a) 70% Ethanol
b) 95% Ethanol
c) 100% Ethanol
d) Xylene

10
Protocol: Formalin-Fixed, Parrafin-Embedded (FFPE) contd.

3. Embed the tissue in molten paraffin. After the paraffin solidifies keep the blocks at 4°C until sectioning.
Note: Paraffin melts at 57°C.

4. Keep FFPE blocks chosen for sectioning tissue face down in an ice water bath, to hydrate the tissue and
avoid cracking during sectioning. Certain tissues (e.g. liver or spleen) require this to be repeated after
10-20 sections.

5. Use a microtome to cut the paraffin tissue blocks into 4-10 μm thick sections and transfer them to a
37°C water bath with distilled water.

6. Pick up the floating tissue section using a clean histological slide (coated with gelatin or poly-L-lysine to improve
adhesion of tissue sections) and allow mounted tissue sections to dry for about 30 min on a 37°C hot plate
followed by baking them for 2-3 hours in a 40°C oven.

Note: Slides can be safely stored at room temperature until ready for staining. Storage of cut slides for
longer than 1 month is not recommended.

Protocol: Method I - Frozen Samples (Whole Animal or Dissected Tissue with Aldehyde/Formalin Fixation)

1. Fixation:

Whole animal - Perfuse the animal with warm saline solution to flush the blood out of vasculature and
immediately follow this by perfusion with freshly prepared 4% formaldehyde.

Dissected Tissue - Immerse tissue in 4% formaldehyde (50-100x the tissue volume) for 4-24 hours at room
temperature. Wash tissue with PBS.

Note: Fixation temperature and time may require optimization depending on the tissue type and size.

2. Cryoprotection:

Whole animal - Continue perfusion with a 10% sucrose solution.

Dissected Tissue - Immerse dissected tissue in a 10% sucrose solution overnight at 4°C.

Note: Tissue will sink in sucrose solution upon reaching equilibration.

3. Embed tissue in OCT cryostat sectioning medium and store at -80°C until ready for sectioning.
Note: Tissue can be safely stored for up to 1 month.

4. When ready for sectioning, move the embedded tissue directly into the cryostat and use OCT medium to
mount it to the chuck. Allow the temperature of the tissue to equilibrate with the cryostat. Cut the tissue in
5-20 μm thick sections. Mount tissue sections onto gelatin or poly-L-lysine coated slides by placing the cold
sections onto warm slides.

Note: Tissue can be safely stored for up to 1 month.

11
 Protocol: Method II - Cryopreservation & Sectioning of Alcohol Fixed Tissues

1. After dissection, immediately snap freeze tissue with isopentane cooled by dry ice. To do this, fill the styroform
box with dry ice and pour isopentane over it so that chunks of dry ice are halfway immersed in isopentane. Place
a metal cryostat chuck on top of the dry ice/isopentane slurry and then mount piece of tissue on top of the chuck
and cover it with OCT.

2. Wait until tissue in OCT is completely frozen (OCT turns from clear to opaque white solid material) and
transfer chuck with frozen tissue into the cryostat. Allow the temperature of the tissue to equilibrate to the
temperature in the cryostat.

3. Cut the tissue in 5-20 μm thick sections. Mount tissue sections onto gelatin or poly-L-lysine coated slides:
place tissue sections onto cold slides and then warm the slides up to thaw tissue sections so that they can
permanently adhere to the slides.

Note: Slides can be safely stored for 6-12 months at -80°C until ready for fixing. Uncut tissue
can be re-stored at -80°C.

4. Remove slides from freezer and fix with cold fixative (acetone or methanol) for 10 minutes. Proceed to staining.

How to Perform Immunostaining on Organoids

Organoids are 3D miniature versions of organs that are
generated in vitro and can be derived from either tissue
or stem cells. Due to their unique 3D nature, specific
protocols have been designed to preserve morphology
and analyze marker expression via immunostaining.

View Protocol for Harvesting, Fixing,

and Immunostaining Organoids

12
IHC
WORKFLOW
(Epitope)
Antigen
Retrieval

13
(Epitope) Antigen Retrieval

Formalin is a superior fixative for preserving tissue What is Antigen Retrieval?

morphology, but it also adversely impacts IHC staining
by masking epitopes and restricting antibody-target Antigen retrieval is a step in the IHC process that allows
binding. Masking is the result of crosslinks created between the removal of crosslinks created by formalin fixation.
amino acids both within the target antigen and between Proteolytic-Induced Epitope Retrieval (PIER) and Heat-
surrounding proteins. Crosslinks can impede an antibody Induced Epitope Retrieval (HIER) are two of the most widely
from effectively accessing its epitope, limiting the ability to used antigen retrieval methods for FFPE tissue sections. PIER
detect antigens in formalin-fixed tissue samples. This results is a method of antigen retrieval which relies on enzymes
in a weak signal or a signal that is indistinguishable from such as proteinase K, trypsin, or pepsin to unmask antigen.
the background. Masked epitopes can be recovered with HIER utilizes heat to promote epitope availability. Because
an antigen retrieval step, which works to promote epitope ideal retrieval conditions are influenced by the tissue type
availability and enhance antigenicity. and fixation method, optimization of the retrieval protocol
for each antigen is highly recommended.

Heat-Induced Epitope Retrieval (HIER) Proteolytic-Induced Epitope Retrieval (PIER)

The use of heat to retrieve antigen The use of enzymes to retrieve antigen
What is it?
and restore antigenicity and restore antigenicity

How does it work? Heat causes crosslinked protein to unfold Enzymes degrade protein crosslinks

Tissue Prep FFPE and Frozen tissues FFPE tissues

Recommendation First choice Harsher condition, favored for heavily crosslinked samples

No HIER Acidic HIER Figure 6. Antigen Retrieval Improves IHC Detection of p27.
Immunohistochemical images show the detection of p27 in
paraffin-embedded human prostate cancer sections following incubation
of tissue for 10 minutes at 95 °C in the specified antigen retrieval solution.
Compared to no HIER treatment, p27 detection was enhanced following
incubation in neutral (pH 7.0) and basic (pH 9.5) HIER solution but not the
acidic (pH 5.0) antigen solution. P27 was detected using Rat Anti-Human/
Mouse/Rat p27/Kip1 Monoclonal Antibody (brown; R&D Systems Catalog
#MAB22561) Image from R&D Systems: Antigen Retrieval Methods.

VisUCyte Antigen Retrieval Reagents: Basic (R&D Systems Catalog #VCTS021),

Acidic (R&D Systems Catalog #VCTS022), Universal (R&D Systems
Neutral HIER Basic HIER Catalog #VCTS023)

14
Is Antigen Retrieval Always Necessary?

Antigen retrieval is recommended for most FFPE tissues. However, not all IHC experiments require a retrieval step. Some targets
are negatively impacted by it and antigen retrieval on frozen tissue is not recommended. The fixation method and duration, in
addition to the type of tissue, target antigen, and type of antibody determine the need for antigen retrieval:

• The antigen retrieval process can be too harsh and damage the tissue.

• A polyclonal antibody may enhance detection of antigen compared to a monoclonal due to its ability to
bind multiple epitopes.
• A change in pH or cation concentration of antibody diluent or a simple change in the incubation conditions of primary
antibody can also improve antibody affinity for its epitope without the need for a formal antigen retrieval step.

Protocol: Heat-Induced Epitope Retrieval (HIER)

Select one of the following HIER buffer options:

a) Citrate Buffer - 10mM Citric Acid, 0.05% Tween 20, pH 6.0

(also see: Pre-mixed Citrate Buffer from Novus Biologicals Catalog #NB900-62075)
b) Tris Buffered Saline (TBS) - 50mM TBS, 0.05% Tween 20, pH 9.0
c) EDTA Buffer - 1mM EDTA, 0.05% Tween 20, pH 8.0
d) VisUCyte™ Antigen Retrieval Reagents

1. Pre-heat retrieval solution in a staining dish in a vegetable steamer until temperature reaches 95-100°C.
Note: A microwave or pressure cooker can be used as alternative heating source in place of the steamer.

2. Immerse slides in the staining dish.

3. Place the lid loosely on the staining dish and incubate it for 20-40 minutes in the steamer.

4. Remove the staining dish from the steamer and place it on the lab bench at room temperature.

5. Allow the slides to cool for 20-30 minutes before proceeding with the staining procedure.

Protocol: Proteolytic-Induced Epitope Retrieval (PIER)

Select one of 2 PIER buffer options:

a) Trypsin Working Solution, 0.05%

b) Proteinase K Working Solution, 20 μg/ml

1. Cover sections with chosen PIER buffer.

2. Incubate for 10-20 minutes at 37°C in humidified chamber.

3. Allow sections to cool at room temperature for 10 minutes.

15
IHC
WORKFLOW
Blocking
Non-Specific
Binding

16
Blocking Non-Specific Binding
Antibody-based applications rely on the specific binding of Page 19 further describes tissues commonly associated with
antibody to the target epitope to generate accurate expression high non-specific binding, the affected step or detection
data. Antibodies specifically recognize the epitope of interest reagent, additional steps to minimize non-specific signal, and
through intermolecular forces including hydrophobic proper background controls.
interactions, ionic interactions, hydrogen bonding, and
others. However, the same forces that govern specific
interactions can also contribute to non-specific binding, i.e.
binding of the primary antibody to amino acids other than
those within the desired epitope of the antigen. The challenge
Tip: The detection method should be compatible with
in IHC experiments is to reduce non-specific interactions
the sample tissue. For detecting a low abundance
without impairing specific antibody-epitope recognition.
protein in tissues with high levels of endogenous
Causes of non-specific staining include interactions of biotin, consider using a polymer-based signal
the primary and secondary antibodies with serum proteins, amplification method.
ionic interactions between antibodies and tissues, and
interactions with endogenous molecules capable of affecting
the IHC detection system used. These issues can result
in high background and an inability to visualize the
antigen of interest in its appropriate cellular location.
Staining problems of this type can be addressed by
blocking non-specific interactions using a blocking reagent.
Blocking other reactive epitopes and quenching endogenous
enzymatic reactions prior to primary antibody incubation
prevents non-specific binding and mitigates false positive
staining (i.e., incorrect identification of positive signal).

The choice of blocking buffer is also contingent on the

method of detection used. For instance, if using an
alkaline-phosphatase (AP) conjugated secondary antibody,
No Blocking + Blocking
the blocking serum should be diluted in Tris buffered
saline (TBS). PBS will interfere with the alkaline
phosphatase reaction.

Common buffers to block non-specific interactions are serum,

BSA, casein, or commercial buffers. If blocking with the
serum, the host of the secondary antibody should be used to
determine the species of the animal serum. For example, if
the secondary antibody is goat anti-mouse, use goat serum as
Figure 7. Blocking Non-specific Binding with Serum.
a blocking agent. (Left) CD14 was detected in paraffin-embedded human tonsil tissue using
anti-human CD14 biotinylated affinity-purified polyclonal antibody (Catalog #
Autofluorescence is the endogenous level of fluorescence BAF383). Tissue was subjected to antigen retrieval using the Basic Antigen
Retrieval Kit (Catalog # VCTS021). Tissue was stained using high sensitivity
emitted by tissues when examined under fluorescence streptavidin conjugated to HRP (HSS-HRP) and DAB, and counterstained with
microscopy. Autofluorescence can impact imaging for hematoxylin (blue). (Right) Non-specific background staining is markedly
reduced in a parallel experiment which included a blocking step using animal
immunofluorescence (IF) detection, especially for tissue serum for 15 minutes at room temperature prior to incubation with the
samples with elevated levels of flavins or porphyrins. primary antibody.
17
A B
Figure 8. Quenching Endogenous Peroxidase Activity.
(A) Failing to quench endogenous peroxidase prior to staining produced a
false positive signal in sections of human kidney. Tissue was stained using an
Anti-Goat HRP-DAB Cell & Tissue Staining Kit (brown). (B) Endogenous
peroxidase activity was quenched in the same tissue using 3% H2O2 in
methanol for 15 minutes at room temperature prior to staining.

18 18
Endogenous Activity
and Reactive Epitopes
Endogenous Activity Reactive Epitopes

Peroxidase Biotin
• Tissues with High Activity - Kidney, liver, or vascular • Reactive Tissues/Conditions - Liver, kidney, spleen,
areas with red blood cells, lysosomal membranes heart, brain, and lung or frozen tissues
especially in active phagocytic cells
• Affected Step - Chromogenic detection using biotin
• Affected Step - Chromogenic detection with HRP conjugated reagents

• Block - Treat tissue with 3-10% hydrogen peroxide in • Block - Treat tissue with avidin prior to incubation with
methanol prior to incubation with HRP conjugated biotin-conjugated reagents. Then treat sample with biotin
secondary antibody. to block additional biotin binding sites on the
avidin molecule.
• Background Control - Before the antibody incubation
step, incubate the sample with DAB substrate. The • Background Control - Before the antibody incubation
presence of endogenous peroxidase will be associated step, incubate the sample with avidin-biotin complex
with the deposition of brown color. or streptavidin (SA)-HRP, then DAB. The presence of
endogenous biotin will be associated with the deposition
of brown color.

Autofluorescence
Phosphatase
• Tissues/Conditions with Endogenous Fluorescence -
• Tissues with High Activity - Intestine, kidney, lymphoid Pancreas, brain, red blood cells, other pigmented cell
types, lipofuscin, extracellular matrix components, or
• Affected Step - Chromogenic detection with AP
formalin-fixed
• Block - Treat tissue section with 1mM Levamisole prior to
• Affected Step - IF detection, most severe in the shorter
incubation with AP conjugated secondary antibody. For
visible fluorescence wavelengths
intestinal sections, block with 1% acetic acid.
• Block - For aldehyde autofluorescence, block with sodium
• Background Control - Before the antibody incubation
borohydride in PBS. Choose fluorochromes emitting at
step, incubate the sample with nitro blue tetrazolium/5-
unaffected wavelengths.
bromo-4-chloro- 3-indolyl phosphate (NBT/BCIP)
substrate. The presence of endogenous phosphatases will • Background Control - Prior to staining, examine tissue
be associated with the deposition of blue color. sections under fluorescent microscope using appropriate
filter sets.

19
IHC
WORKFLOW
Antibodies
& Detection
Methods

20
Antibodies & Detection Methods
One of the most important decisions that can affect the require a PTM such as phosphorylation to become activated,
outcome of an IHC experiment is selecting an appropriate and to distinguish between inactive and activated AKT an
primary antibody. It is critical to choose a high-quality antibody that specifically recognizes this activated form
primary antibody that specifically binds the target antigen. A should be used, e.g. p Ser473.
target protein’s function, tissue, and subcellular localization,
along with any post-translational modifications (PTMs) In addition to antibody specificity, the clonality of the
should be taken into consideration when choosing the antibody should also be considered. Antibodies can be either
appropriate antibody. For example, some proteins, e.g. AKT1, monoclonal or polyclonal.

Antibody Clonality: Polyclonal vs. Monoclonal

Polyclonal Antibody Monoclonal Antibody Recombinant Antibody

Antibodies generated from Antibodies generated from A type of monoclonal antibody

Antibody Production multiple B cell clones a single B cell clone generated in vitro using
recombinant DNA technology

Multiple epitopes from A single epitope A single epitope

Epitopes Recognized
the same antigen

· Antigen-antibody binding is · Less lot-to-lot variability · Highest reproducibility and

less affected by changes to · Reduced non-specific binding, lot-to-lot consistency
antigen conformation from thus lower background staining · Ability to be engineered, such
Advantages sample preparation and fixation as a species scaffold swap
· Binding to multiple epitopes · Less production time compared
can enhance signal to monoclonal antibodies

· Higher background · Less tolerant to changes in · More costly development

Disadvantages · Higher lot-to-lot variability antigen conformation from
fixation or processing

Typical Working Concentration range: Concentration range: Concentration range:

Conditions 1.7-15 µg/mL 5-25 µg/mL 5-25 µg/mL

If using an antibody already validated for IHC, a basic staining protocol may be available. Keep in mind that experimental
conditions such as tissue type, species and sample preparation may differ and should all be factored in to establish the optimal
staining protocol. The best antibody concentration, diluent, and incubation time should be determined to maximize the specific
signal while minimizing the contribution to non-specific staining. A good starting point is to vary the antibody concentration
and keep the incubation time constant (overnight at 4 °C).

21
If using an antibody already validated for IHC, a basic Polyclonal Antibody Monoclonal Antibody
staining protocol may be available. Keep in mind that
experimental conditions such as tissue type, species, and
sample preparation may differ and should all be factored
in to establish the optimal staining protocol. The best
antibody concentration, diluent, and incubation time
should be determined to maximize the specific signal while
minimizing the contribution to non-specific staining. A good
starting point is to vary the antibody concentration and keep
the incubation time constant (overnight at 4 °C).
Figure 9. Polyclonal vs Monoclonal Antibody Binding.
Polyclonal Antibodies (left) are capable of recognizing multiple epitopes
Why Choose a Polyclonal Antibody from the same antigen whereas Monoclonal Antibodies (right) only
recognize a single epitope.
for my IHC Experiment?
Antibody binding for IHC/ICC experiments is often
dependent on the target protein maintaining its native
conformational state. A change in protein from its native
state can impact antibody-epitope binding and affect IHC
staining. Access to a desired epitope can be compromised
by interactions with other proteins, post-translational
modifications, temperature, pH, fixation, and salt
concentration. Because polyclonal antibodies are capable of
binding multiple epitopes of the same antigen, the staining
result is less likely to be affected by changes in the target
protein conformation state than monoclonal antibodies.
In general, polyclonal antibodies are also more stable
than monoclonal antibodies over a range of pH and salt
concentration. Hence, polyclonal antibodies are often used
for IHC/ICC experiments.

22
Detection Methods

Following incubation with the primary antibody, antigen targets. TSA utilizes HRP’s catalytic activity for in situ
expression is visualized using an appropriate detection labeling of the target protein or nucleic acid sequences.
system. The method of detection can be direct or indirect The basis for boosting signal amplification comes from the
and may generate a fluorescent or chromogenic signal. specific and high-density deposition of TSA dyes adjacent to
the HRP-labeled antibody probe.
Direct vs. Indirect Direction
These signal amplification methods, along with direct
Direct detection involves the use of primary antibodies and indirect detection, are described further in the table
that are directly conjugated to a label. Frequently, IHC on the next page.
uses the indirect method of detection in which a secondary
antibody, directed against the constant region of the primary
antibody, carries the label. The indirect method is more
sensitive than using a directly labeled primary antibody
because multiple labeled secondary antibodies can bind to
a single primary antibody.

Fluorescence vs. Chromogenic Signal

Both direct and indirect detection methods can be

visualized by either immunofluorescence (IF) or by a
chromogenic reaction. In IF detection, the fluorochrome
conjugated antibody is excited by and emits light at specific
wavelengths. In chromogenic detection, the antibody is
conjugated to an enzyme (such as HRP) which converts
Figure 10. Example of Fluorescent IHC Detection.
DAB or 3-amino-9-ethylcarbazole (AEC) to a colored CD31/PECAM 1 was detected in immersion fixed frozen sections of mouse
precipitate at the antigen site. embryo using Goat Anti-Mouse/Rat CD31/PECAM 1 Antigen Affinity-purified
Polyclonal Antibody (R&D Systems Catalog # AF3628). Tissue was stained
using the NorthernLights™ 557-conjugated Anti-Goat IgG Secondary
Signal Amplification Antibody (yellow; R&D Systems Catalog # NL001) and counterstained with
DAPI (blue).

For detection of low abundance antigens, additional steps

to amplify the antigen signal may be required. Two
particular signal amplification methods for chromogenic
detection make use of the high affinity complex between
biotin and SA (or avidin). Biotin conjugated secondary
antibodies link tissue-bound primary antibodies with either
an avidin-biotin-peroxidase complex in the avidin-biotin
complex (ABC) method or a SA-peroxidase complex for the
labeled SA-biotin (LSAB) method. Since ABC and LSAB
complexes exhibit a high enzyme-to-antibody ratio, these
are more sensitive methods compared to the traditional
indirect method.

Unlike ABC and LSAB, the polymer-based detection method

is biotin-free. This third method of signal amplification uses Figure 11. Example of Chromogenic IHC Detection.
beta -III Tubulin was detected in immersion fixed paraffin-embedded sections
a polymer backbone containing multiple enzyme molecules of human brain using Mouse Anti-Neuron-specific beta -III Tubulin
(e.g. HRP) and is directly conjugated to the secondary Monoclonal Antibody (R&D Systems Catalog # MAB1195) followed by
incubation with the Anti-Mouse IgG VisUCyte™ HRP Polymer Antibody (R&D
antibody. In addition to chromogenic signal amplification, Systems Catalog # VC001). Tissue was stained using DAB (brown) and
tyramide signal amplification (TSA) is a highly sensitive counterstained with hematoxylin (blue).

method that can be used during either chromogenic or

fluorescent IHC to enhance the detection of low-abundance

23
 Signal Amplification Methods

Direct Indirect Biotin-Conjugated Polymer-Based TSA

Format

· Quick · Greater sensitivity · Greater sensitivity · Greater sensitivity · Suitable for

· Less steps than · More flexibility compared to compared to ABC fluorescent IHC
indirect detection compared to traditional indirect and LSAB method · Multiplexing
detection · Fewer steps
· Good for direct detection capabilities
Advantages multiplexing because it’s
· LSAB smaller than ABC and
complex size over LSAB method
possible to
ABC facilitates
use different
better tissue
conjugated
penetration
secondaries

· Lower sensitivity · Potential · Presence of · Larger size · Can be

· Selection non-specific endogenous with polymer more costly
of directly binding of biotin in tissues backbone may · Possible increase
secondary can increase limit tissue in background
Limitations conjugated
antibodies background penetration
antibodies can staining
be limited
IHC Staining: IF and Chromogenic

Protocol: Deparaffinization & Rehydration for Paraffin-Embedded Tissues (FFPE Tissue Sections)

1. Paraffin wax must be removed from FFPE tissue sections, and the tissue must be rehydrated before antibody
staining. Xylene is the traditional organic solvent for removing paraffin.

Deparaffinize and rehydrate by immersing the slides with:

a) Xylene, three washes – 5 minutes each
b) 100% Ethanol, two washes – 10 minutes each
c) 95% Ethanol, two washes – 10 minutes each
d) 70% Ethanol, two washes – 10 minutes each
e) 50% Ethanol, two washes – 10 minutes each
f) Deionized Water, two washes – 5 minutes each

Note: Before moving to alcohol grades step, make sure to completely deparaffinize the sections. If the
sections still have traces of wax, immerse in Xylene for an extra 5 minutes.

DO NOT let the tissue dry from this point on.

Protocol: IHC Staining

Note: For IHC-Fr sections, warm slides to roomtemperature and wash slides 2x with PBS.

1. Apply an antigen retrieval protocol as needed for formaldehyde-fixed tissue sections (see page 15 for protocol).

2. Draw a circle on the slide around the tissue with a hydrophobic barrier pen (eg, PAP pen).

3. Block for endogenous activity or reactive epitopes as needed (see page 17).
Note: For chromogenic detection, if using HRP reagents, block endogenous peroxidase activity by quenching
the tissue sections with 3.0% hydrogen peroxide in methanol for at least 15 minutes. Afterwards, wash the
sections by immersing them in distilled water for 5 minutes.

Optional Permeabilization & Intracellular Staining

Permeabilization of membranes is required for antibodies to access the inside of a cell for staining of
intracellular antigens. Some fixatives, such as acetone and methanol, also act as permeabilization agents and
IHC protocols using these fixatives may not require this additional step. Two potential permeabilization steps
are listed below:

Option 1: Incubate slides in 0.1% Triton or NP-40 in PBS for 10 minutes.

Option 2: Incubate slides in 0.2 to 0.5% Tween 20, Saponin, Digitonin, or Leucoperm in PBS for 10 to 30 minutes.

The appropriate use of a permeabilization agent depends on the type of fixative, tissue size, and the cellular
location of the antigen.

4. Block any non-specific binding by incubating the tissue sections with 5% animal serum in
PBS + 0.3% Triton X-100 (PBS-T) for 30 minutes at room temperature.

25
 Protocol: IHC Staining contd.

5. Add the primary antibody diluted in 1% animal serum in PBS-T and incubate at room temperature for 1-2 hours.
Continue the incubation overnight at 4°C in a humidified chamber.

Reminder: The host species of the primary antibody should differ from the source of the tissue sample.

Note: Use the recommended dilution of the antibody specified on the datasheet. If not specified,
use 3-fold serial dilutions: 2 μg/mL, 6 μg/mL and 18 μg/mL.

6. Wash sections twice with 1% animal serum in PBS-T for 10 minutes each.

7. Proceed with either the IF or chromogenic staining protocol.

Protocol: IF Staining

1. Add a fluorochrome-conjugated secondary antibody and incubate at room temperature for 1 -2 hours.
Use the recommended dilution of the antibody as specified on the data sheet.

Note: The secondary antibody should be directed against the host species of the primary antibody. For help on
selecting secondary antibodies, see our Secondary Antibody Handbook.

2. Wash sections twice with 1% animal serum PBS-T for 10 minutes each.

3. For nuclear staining, add DAPI solution (~2.9 μM) or other DNA binding dye (e.g. Hoechst 33342) and incubate
2-5 minutes at room temperature. Rinse 1x with PBS. DAPI has an absorption maximum at 358 nm and an
emission maximum of 461 nm.

4. Apply a drop of mounting media containing a fluorescence anti-fade agent. Carefully place a coverslip on it
and remove the excess mounting media, if necessary. Circle the edges of the coverslip with clear fingernail
polish to prevent the cells from drying. Allow nail polish to air dry.

Note: Some mounting media solutions have DAPI already added and will harden after exposure to air, eliminating
the need to seal the edges of the coverslip.

5. Examine the cells under a fluorescence microscope using the appropriate excitation and emission filters and
image as required. Slides can be stored between -20°C and 4°C in a dark slide box or slide book.

Reminder: When examining slides using a fluorescence microscope avoid long exposures at the fluorochrome’s excitation
wavelength to prevent photo-bleaching.

26
Protocol: Chromogenic Staining (ABC amplification method)

1. Add a biotin conjugated secondary antibody and incubate at room temperature for 1 hour. Use the
recommended dilution of the antibody as specified on the data sheet.

Note: The secondary antibody should be directed against the host species of the primary antibody.
For help on selecting secondary antibodies, see our Secondary Antibody Handbook.

2. Wash sections twice with 1% animal serum PBS-T for 10 minutes each.

3. Add ABC-HRP reagent and incubate at room temperature for 1 hour. Follow manufacturer’s guidelines
for reagent preparation.

4. Prepare a working solution of DAB and apply to tissue sections. Monitor the reaction as the chromogenic
reaction turns the epitope sites brown (time of color development may vary from few seconds to 10 minutes).
Proceed to the next step when the intensity of the signal is appropriate for imaging.

Reminder: DAB is a carcinogen. Always wear gloves and work in a fume hood when working with DAB. Deactivate and clean
work area after use according to manufacturer’s instructions.

5. Wash sections twice in PBS for 10 minutes each.

6. Wash the sections twice in distilled water for 2 minutes each.

7. To counterstain nuclei, use Hematoxylin according to the manufacturer’s instructions.

Note: If using an aqueous chromogen instead of DAB (i.e. AEC, Fast Red, etc.), skip the following dehydration
step and mount in aqueous media instead of organic mounting media.

8. Dehydrate tissue sections by moving slides through the following solutions twice for 2 minutes each:
a) 95% Ethanol
b) 100% Ethanol
c) Xylene

9. Add mounting media to slides and top with coverslips. The DAB reaction is permanent and stable and can be
analyzed under a bright-field microscope at any time.

IHC Controls

Due to the number of variables in the protocol that can A distinct benefit of IHC is visually being able to detect or
introduce artifacts or affect IHC staining, inclusion of “see” your protein of interest and, more specifically, the
appropriate controls in each experiment is important for cells or subcellular compartments expressing that target.
identifying the source of potential staining issues and for In addition to using controls, resources and tools such as
accurate interpretation of results. A good experimental UniProt, The Human Protein Atlas, and antibodies cited in
design produces results that demonstrate that the antigen publications can all be used for analysis to help gauge where
is localized to the correct specialized tissues, cell types, or to expect target expression and localization.
subcellular location.

27
Tissue Type Controls

Controls for IHC/ICC experiments include using tissue samples that are known to express (or not express) the epitope of interest.
This strategy can provide a useful reference and may also be utilized during initial optimization studies. Tissue samples from
different species can be included to support the species specificity of an antibody.

Tissue Control What it Tells You How it Helps Commonly Used

Confirms the presence target Ensures the IHC protocol itself is · Tissue or experimental
antigen working properly, even if experi- condition with proven
mental samples are negative positive signal
Positive · Samples from
transgenic animals
that overexpress
the antigen

Does not express the target antigen Determines the degree of back- · Tissue or experimental
ground staining caused by condition with proven
non-specific interactions. Back- negative signal
Negative ground staining should be · Samples from
negligible and not resemble knockdown or
specific staining.
knockout tissues

Immunoreactivity Indicates significant damage to Vimentin- stains mesenchymal and

quality control tissue antigens, tissue is over-fixed endothelial cells and is an internal
Tissue Integrity
or other failure of the staining quality control for antigenicity
procedure

View Tissue Control

Slides From Novus

Tissue Artifact/Endogenous Tissue A. B. C.

Background Control

Background staining may be more pronounced in particular

tissues and thus it is important to use an endogenous tissue
background, or tissue artifact, control. Since high background
can mask positive signal from low abundance antigens or
lead to artifacts being mistaken for specific staining, IHC Figure 12. Lipofuscin Background in the nervous system.
results may be misinterpreted. Before applying primary Lipofuscin has autofluorescent properties that overlap with the excitation
and emission spectra of commonly used fluorochromes. Circled in the
antibodies, tissues should be examined under a bright-field micrographs above are Lipofuscin-containing neurons that may appear
or fluorescence microscope (for chromogenic or fluorescent labeled using either bright-field microscopy (A) or fluorescence microscopy
in the green (B) and red (C) spectrums.
labels, respectively) to ensure the signal is not due to
the inherent properties of the tissue itself. For example,
lipofuscin is an endogenous autofluorescent pigment that
accumulates in lysosomes of postmitotic cells and may
be confused with positive staining for both bright-field
and fluorescence microscopy. Biological autofluorescence,
especially in human tissues, is common with aging.
Antibody Controls

The specificity of the antigen-antibody interaction is critical for accurate detection of the antigen. Due to the complex nature
of tissue and the multifactorial effect of sample preparation on the staining procedure, antibody controls help to confirm the
presence of the antigen of interest. A few recommended antibody controls include:

No Primary Antibody Control: The primary antibody is absent from the diluent during the primary antibody incubation
step. This control will show the contribution of all other components, such as the secondary
antibody and detection reagents, to background staining (Figure 13).

Isotype Control:
Should be used to confirm the specificity of the primary antibody. For this control, the
primary antibody should be replaced with a non-immune immunoglobulin of the same
isotype and at the same concentration as the primary antibody. Example isotypes are IgG1,
IgG2a, IgG2B, and IgM. All other conditions and protocol steps should remain the same. This
control will demonstrate if the staining by the primary antibody is a result of an interaction
with the antigen binding site (paratope) or due to non-specific interaction with the
immunoglobulin molecule.

Absorption Control: Also used to demonstrate that an antibody binds specifically to the antigen of interest. For
this control, the primary antibody is first inactivated by preincubation with the immunogen
(antigen) and then used in place of the primary antibody in the IHC protocol. The antigen to
antibody solution should be prepared at a molar ratio of 10:1 to fully saturate the antibody
and then incubated in the IHC antibody diluent overnight at 4°C. The staining pattern
produced by the primary antibody can be compared to that produced by the
pre-absorbed antibody.

The optimal immunogen for absorption is small, purified peptide. With antibodies raised
against the whole protein, addition of the antibody plus protein may result in higher
non-specific staining. This is due to the antigen itself binding to the tissue. Thus, it is
important to note that an absorption control using whole protein may not always confirm
the specificity of an antibody and results should be analyzed with discretion.

+ Primary Antibody No Primary Antibody A B

Figure 13. Primary Antibody Control in Human Kidney. Figure 14. Absorption Control in Rat Dorsal Root Ganglion.
Neprilysin/CD10 was detected in a FFPE section of human kidney using Goat (A) A cryostat section of rat dorsal ganglion stained for phospho-MSK1
Anti-Human Neprilysin/CD10 Polyclonal Antibody (R&D Systems Catalog # (S212) using Anti-Human phospho-MSK1 Affinity-Purified Polyclonal Antibody
AF1182). Tissue was stained with an anti-Goat HRP-DAB Cell & Tissue Staining (R&D Systems Catalog # AF1036). (B) Nuclear staining (indicated by arrows)
Kit (brown) and counterstained with hematoxylin (blue). Neprilysin/CD10 is abolished if the antibody is first pre-absorbed with the S212
staining (brown) is lost if the primary antibody is absent from the diluent in phosphorylated immunogen.
a parallel experiment.

29
Visual Workflow
Traditional IHC - Sample Preparation & Fixation

30
Traditional IHC - Sample Preparation & Fixation contd.

31
ISH-IHC (RNA-Protein Co-Detection)

Tissue Cells

RNA 1 RNA 3 RNA 1 RNA 3

RNA 2 RNA 2

Figure 15. Advanced Cell Diagnostic’s (ACD’s) Integrated Co-Detection Workflow (ICW) for simultaneous detection of protein and RNA.
The ICW allows for inclusion of wider range of antibodies to be combined with RNA ISH enabling researchers to acquire more data
and conserve precious samples.

Troubleshooting Guide
Problem: Lack of Staining / No Signal

Possible Cause Explanation Recommendation(s)

Protein isn’t expressed in tissue or under Check protein expression by Western Blot or in situ
Antigen
experimental conditions. hybridization (in some rare cases translation may be
not present
blocked even though mRNA is detected).

Over-fixation can cause epitope masking. Reduce the time or concentration of the fixative.
Apply an antigen retrieval step.

Tissue fixation Under-fixation can cause heavy edge staining Increase the time or concentration of the fixative.
with little to no positive signal in middle of your
specimen. It can also result in autolysis and Alternatively, try a different fixative.
target protein degradation.

Epitope altered during embedding process and not Switch to using frozen sections.
Paraffin-Embedding recognized by primary antibody.
Embed tissue at 58 °C or below.

Ineffective Antigen Epitope masking prevents binding of Increase treatment time or change procedure (either
Retrieval primary antibody. new buffer or different method).

Inadequate penetration of antibodies Use 0.5-1.0% Triton (or Tween-20)

Permeabilization
and buffers into the tissue sections. detergent in the buffers.

Antigen was destroyed before incubation with the If quenching of endogenous peroxidase was
primary antibody. done prior to the addition of primary antibodies,
Antigen Damaged
block peroxidase after incubation with the
primary antibody.

Antibodies do not work due to improper storage. Follow storage instructions on the datasheet. In
general, aliquot antibodies into smaller volumes
Antibody Storage sufficient to make a working solution for a single
experiment. Store aliquots in a manual defrost freezer
(–20 to –70 °C) and avoid repeated freeze-thaw cycles.
 Problem: Lack of Staining / No Signal contd.

Possible Cause Explanation Recommendation(s)

Antibody Epitope isn’t saturated with antibody and/or hasn’t Increase the concentration or incubation time of the
Application reached binding equilibrium. primary or secondary antibody.

Primary antibody isn’t reactive in the tissue sample. Confirm the species reactivity of the
primary antibody.

Antibody Primary antibody binds an epitope only exposed Confirm the antibody can be used for assays in which
Compatibility when protein is unfolded. the protein is in its native conformation.

Incompatible primary and secondary antibody. Verify that the secondary antibody will interact with
the species of the primary antibody.

Missing reagents or added improperly/used in Repeat staining procedure and verify all reagents are
Reagents
wrong order. added in correct order.

Microscope Fluorochrome isn’t effectively excited or emission Verify fluorochrome is compatible with filter sets and
Adjustments isn’t captured by filters. appropriate settings are used. Also, increase the
(Fluorescence) camera exposure time.

No Antigen Staining Good Antigen Staining

33
 Problem: High Background

Possible Cause Explanation Recommendation(s)

Tissue Fixation Over-fixation can cause strong non-specific staining. Reduce the time or concentration of the fixative.

Inadequate blocking. Increase the incubation time or concentration of

Blocking Step
serum in the blocking buffer.

Tissue may have high autofluorescence, Check slides prior to staining and use the
Endogenous
biotin, peroxidase activity, etc. appropriate blocking/quenching steps. Try
Activity/Reactivity
a more compatible detection method.

Primary or secondary antibody concentration Titrate antibodies to find the optimal signal to
too high. background staining.
Antibody
Application Non-specific binding of primary or Increase the amount or incubation period
secondary antibody. of washes. Reduce the secondary antibody
incubation period.

Secondary antibody is non-specifically Select a secondary antibody pre-adsorbed

binding the tissue sample. against the species of the experimental
Antibody
sample. For example, a secondary antibody adsorbed
Compatibility
against mouse immunoglobulin or serum is
recommended when staining mouse tissue.

DAB overstaining. Reduce the time of incubation with DAB chromogen.

Endogenous peroxidases are activating Quench with hydrogen peroxide.

Detection Method the DAB reaction.

Endogenous biotin is activating the SA-biotin Block endogenous biotin.

complex using the ABC signal amplification method.

Reagents sticking to old or poorly prepared slides. Use freshly prepared or purchased slides.

Slide Condition
Tissue dried out. Avoid letting the tissue dry during the
staining procedure.

Background from ionic interactions. Increase the ionic strength of the blocking and
Buffer Condition
antibody buffers.

Spectral Overlap Multiple fluorochromes have overlapping Select compatible fluorochrome conjugates.
(Fluorescence) emission spectra.

High Background Low Background

34
 Problem: Poor Tissue Morphology/Quality

Possible Cause Explanation Recommendation(s)

Tissue section appears torn or folded. Air bubbles Re-cut sections using a sharp blade or adjust
under section. the cutting speed. Ignore damaged areas when
analyzing the results.

Tissue Preparation Thick sections cause poor resolution of Cut thinner tissue sections.
tissue morphology.

Frozen sections - Ice crystals may have Repeat procedure or try paraffin
destroyed morphology. embedded procedure.

Under-fixation can lead to tissue damage. Increase fixation time or fixative/tissue ratio.
Try alternative fixing reagent.

Under-fixation leads to tissue sections falling off Increase the fixation time or use alternative fixative.
Tissue Fixation
slides (more common with frozen sections). Use freshly prepared, adequately charged slides.

Autolysis of tissue leading to staining of Increase the fixation time, ratio. Consider using
necrotic debris. cross-linking fixative.

Methods are too harsh and damage tissue. Determine optimal conditions (buffers and
Antigen Retrieval
incubation periods). Try a different method.

Poor Tissue Morphology Good Tissue Morphology

Problem: Uneven or Inappropriate Staining

Possible Cause Explanation Recommendation(s)

Tissue Preparation Thickness of tissue section is variable. Re-cut sections using a sharp blade.

Under-fixation with alcohols can Increase fixing time. Try a different fixative.
produce staining artifacts.
Tissue Fixation
Delay in fixation causes antigen diffusion. Fix tissues immediately. Try a cross-linking
fixative over an organic (alcohol) fixative.

Deparaffinization Incomplete removal of paraffin wax. Use fresh xylene.

Antibody Compati- Primary antibody may bind epitopes on other Switch from a polyclonal to a monoclonal
bility antigens. antibody. Try a different monoclonal antibody.

Cell membrane damage and membrane Use detergent at a lower concentration or apply a less
Permeabilization
proteins removed. stringent detergent (Tween-20).

Tissue dried out. Avoid letting the tissue dry during the
Slide Condition
staining procedure.
Basic Buffer & Reagent Recipes
Sample Preparation & Fixation

10X PBS
For 1 L: 10% Sucrose
For 100 mL:
80 g NaCl 10 g Sucrose
2 g KCl 10 mL 10X PBS
11.5 g Na2HPO4 -7H2O Add Distilled water up to 100 mL
2 g KH2PO4 Mix to dissolve
Add Distilled water up to 1000 mL Filter Sterilize
Mix to dissolve
Adjust pH to 7.4 with 1N NaOH

4% Formaldehyde For 1 L:
100 mL 10X PBS
700 mL Distilled water
Add PBS and water to a beaker on a
stir plate in a ventilated hood. Heat
while stirring to 60°C.
40 g paraformaldehyde powder
Add 1 N NaOH dropwise to raise pH
until the solution clears
Cool and filter sterilize
Add Distilled water up to 1000 mL
Adjust pH to approximately 6.9 with
small amounts of dilute HCl

Antigen Retrieval Buffers

HIER Buffers PIER Buffers

• Citrate Buffer (10mM Citric Acid pH 6.0) • Trypsin Working Solution, 0.05%
1.92 g Citric acid (anhydrous) 1 mL Trypsin stock solution (0.5%)
Add Distilled water up to 1000 mL 1 mL Calcium chloride stock solution (1%)
Mix to dissolve Add Distilled Water up to 8 mL
Adjust pH to 6.0 with 1N NaOH Adjust pH to 7.8 with 1N NaOH
• TBS (50mM TBS pH 9.0) • Proteinase K Working Solution, 20 μg/mL
6.1 g Tris 1 mL Proteinase K Stock Solution (20X)
8.8 g Sodium Chloride (NaCl) 19 mL TE Buffer, pH 8.0. Mix well
Add Distilled water up to 1000 mL
Mix to dissolve
Adjust pH to 9.0 with HCl. Mix well.

• EDTA Buffer (1mM EDTA, pH 8.0)

0.37 g EDTA
Add Distilled water up to 1000 mL
Mix to dissolve
Adjust pH to 8.0 using 1N NaOH
Mix well. Store this solution at room temperature
References
Ascoli CA, Aggeler B. Overlooked benefits of using polyclonal antibodies. Biotechniques. 2018;65(3):127-136. https://doi.
org/10.2144/btn-2018-0065

Childs GV. History of Immunohistochemistry. In: McManus LM, Mitchell RN, eds. Pathobiology of Human Disease. Elsevier,
San Diego; 2014:3775-3796.

Hewitt SM, Baskin DG, Frevert CW, Stahl WL, Rosa-Molinar E. Controls for immunohistochemistry: the Histochemical Society's
standards of practice for validation of immunohistochemical assays. J Histochem Cytochem. 2014;62(10):693-697. https://doi.
org/10.1369/0022155414545224

Hickey JW, Neumann EK, Radtke AJ, et al. Spatial mapping of protein composition and tissue organization: a primer for
multiplexed antibody-based imaging. Nat Methods. 2022;19(3):284-295. doi:10.1038/s41592-021-01316-y

Jensen E. Technical review: In situ hybridization. Anat Rec (Hoboken). 2014;297(8):1349-1353. https://doi.org/10.1002/ar.22944

Packer D. The history of the antibody as a tool. Acta Histochem. 2021;123(4):151710. https://doi.org/10.1016/j.acthis.2021.151710

Stack EC, Wang C, Roman KA, Hoyt CC. Multiplexed immunohistochemistry, imaging, and quantitation: a review, with an
assessment of Tyramide signal amplification, multispectral imaging and multiplex analysis. Methods. 2014;70(1):46-58. https://
doi.org/10.1016/j.ymeth.2014.08.016

Tan WCC, Nerurkar SN, Cai HY, et al. Overview of multiplex immunohistochemistry/immunofluorescence techniques in the era
of cancer immunotherapy. Cancer Commun (Lond). 2020;40(4):135-153. https://doi.org/10.1002/cac2.12023

Tsutsumi Y. Pitfalls and Caveats in Applying Chromogenic Immunostaining to Histopathological Diagnosis. Cells.
2021;10(6):1501. https://doi.org/10.3390/cells10061501

Additional Resources
Kalyuzhny AE. Immunohistochemistry. Essential Elements and Beyond. Springer; 2016.

Dikshit A, Phatak J, Lu H, Pimentel H, Zong H, Zhang B, Ma XJ, Anderson C. Combining the RNAscope ISH technology with
IHC to spatially resolve RNA and protein targets simultaneously. (2021; Application Note, Advanced Cell Diagnostics, a Bio-
Techne Brand). Retrieved from Bio-Techne website: https://resources.bio-techne.com/bio-techne-assets/images/resources/ish-
ihc-app-note-wr.pdf

Phatak J, Lu H, Wang L, Zong H, May C, Rouault M, Qutaish M, Zhang B, Ma XJ, Anderson C. The RNAscopeTM multiplex in
situ hybridization technology enables the incorporation of spatial mapping and confirmation of gene signatures into single cell
RNA sequencing workflows. (2021; Application Note, Advanced Cell Diagnostics, a Bio-Techne Brand). Retrieved from Bio-
Techne website: https://resources.bio-techne.com/bio-techne-assets/images/resources/mk-51-134-sscrna-seq-app-note.pdf

IHC/ICC Protocol Guide. R&D Systems, a Bio-Techne Brand

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