BT Immunohistochemistry Handbook
BT Immunohistochemistry Handbook
BT Immunohistochemistry Handbook
(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
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
References 37
Additional Resources 37
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.
2
Variables Influencing Experimental Design and Optimization
Variable Factors
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.
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
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.
7
Sample Preparation, Fixation,
and Sectioning
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.
Fixation Prior to embedding into paraffin Performed either before or after cryosectioning
8
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).
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.
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:
Dissected Tissue - Immerse dissected tissue in a 10% sucrose solution overnight at 4°C.
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.
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.
12
IHC
WORKFLOW
(Epitope)
Antigen
Retrieval
13
(Epitope) Antigen Retrieval
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
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.
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.
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.
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.
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).
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.
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.
23
Signal Amplification Methods
Format
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.
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.
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.
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:
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.
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.
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.
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
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.
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.
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Visual Workflow
Traditional IHC - Sample Preparation & Fixation
30
Traditional IHC - Sample Preparation & Fixation contd.
31
ISH-IHC (RNA-Protein Co-Detection)
Tissue Cells
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
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).
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.
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.
33
Problem: High Background
Tissue Fixation Over-fixation can cause strong non-specific staining. Reduce the time or concentration of the fixative.
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.
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.
34
Problem: Poor Tissue Morphology/Quality
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.
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.
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
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
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