Special Stains For The Carbohydrate
Special Stains For The Carbohydrate
Special Stains For The Carbohydrate
Other than routine haematoxylin and eosin stain, various special stains are now essential parts in routine
polyhydroxyketone groups. They are represented by the common formula Cn(H2O)m. The carbohydrates can
They are also further classified depending on their binding with protein and lipid material.
Stain:
• Alcian blue
• Mucicarmine
Periodic Acid-Schiff’s (PAS) Stain: PAS stain demonstrates neutral polysaccharides that are present in
the basement membrane and also secretion of various glands in our body.
https://www.celnovte-bio-tech.com/periodic-acid-schiff-staining-solution-pas/
Alcian Blue: Alcian blue stains acid mucin (in acidic pH 2.5), such as sialomucin and sulphomucin. It
stains mucin of the salivary glands, prostate and large intestine. Alcian blue also stains proteoglycans of
cartilaginous material.
https://www.celnovte-bio-tech.com/alcian-blue-stain/
Combined PAS-Alcian Blue Staining: s Combined use of Alcian blue and PAS in a same section helps to
demonstrate both acidic and neutral mucin in the same section. This is frequently applied in gastrointestinal
biopsy sections.
https://www.celnovte-bio-tech.com/ab-pas-staining-solution/
Mucicarmine Stain: Mucicarmine stain is applied to demonstrate acid mucin in the malignant cells of
https://www.celnovte-bio-tech.com/mucicarmine-staining-kit/
Contact us [email protected]
Reference
Pranab Dey, Basic and Advanced Laboratory Techniques in Histopathology and Cytology (2018): 81-88
MICROWAVE ALCIAN BLUE METHOD
SOLUTIONS:
Staining Procedure:
Staining Results:
Acidic sulfated mucosubstances ------------------------------------- blue
Cryptococcus ------------------------------------------------------- blue
Nuclei---------------------------------------------------------------- red
Comment:
No single staining solution of alcian blue colors all polyanions maximally, but the above
procedure, done at pH 2.5, stains all except the most strongly sulfated mucopolysaccharides, and
is very useful for demonstrating goblet cell mucins in the respiratory tract and in small and large
bowel, as well as various components of skin, heart valves, large arteries and cartilage.
Alcian blue probably find sits greatest usefulness in combination stains such as Alcian blue,
PAS, and the Kreyberg method.
For a more detailed discussion of various uses of alcian blue see: Schenk, E.A. and Mowry,
R.W.: Alcian Blue. J. Histotech., June 1983, pp. 65-69.
When heating the alcian blue solution with microwave irradiation the top portion of the solution
is warmer by 10-15° C than that near the bottom of the Coplin jar. Therefore, in order to equalize
the temperature of the solution the slides are dipped up and down. This assures uniformity of
staining throughout the tissue sections.
The stain may be performed at room temperature by staining in the alcian blue solution for 30
minutes.
References:
McManus, J.F. and Mowry, R.W.: Staining Methods Histologic and Histochemical, New York,
Paul B. Hoeber, 1960, pp. 137-138.
PERIODIC ACID SCHIFF METHOD
HOME LIST
PERIODIC ACID SCHIFF METHOD
Click the Section Headings (Blue) to Expand/Collapse Material
Image Examples:
Diagnostic Application:
SOLUTIONS:
0.5% Periodic Acid Solution
The formula for Lillies Schiff reagent has been modified. This modified solution requires less
basic fuchsin (pararosanilin) to prepare, can be prepared much faster, yields equally good
staining results, and appears to have greater stability than Lillies formula.
Schiff’s reagent after decolorization with activated charcoal may be either water clear or have a
pale straw color. Some believe that the quality of the activated charcoal determines the final
color of the Schiff’s reagent. Fresh, activated charcoal is supposed to result in a water clear
Schiff’s reagent. From our experience this is not necessarily true as we have found that Schiff’s
reagent prepared with different samples of basic fuchsin will not all decolorize completely even
when fresh activated charcoal is used. The final color of the Schiff’s reagent does not appear to
affect the quality or intensity of the Schiff’s reaction.
Schiff’s reagent can be tested by pouring a few drops of the reagent into 10 ml of reagent grade
formaldehyde (37-40%) in a small beaker. If the solution turns reddish purple rapidly, it is
good. If the reaction is delayed and the resulting color deep blue-purple, the solution is breaking
down and should be discarded.
References:
Garvey, W. et al: Combined modified periodic acid-Schiff and batch staining method. J.
Histotechnol. 15:117-120, 1992.
McManus, J.F.A. and Mowry, R.W.: Staining Methods Histologic and Histochemical, New
York, Paul B. Hoeber, 1960, pp. 126-128.
MICROWAVE PERIODIC ACID-SCHIFF
WITH DIASTASE (DiPAS) HOME
LIST
MICROWAVE PERIODIC ACID-SCHIFF WITH DIASTASE
Click the Section Headings (Blue) to Expand/Collapse Material
Image Examples:
Mucin in intestine (upper left), hepatocytes in liver with amyloidosis (upper right),
intracytoplasmic hyaline globules in liver of alpha-1-antitrypsin deficiency (lower left), and
coccidioides in lung (lower right). Small intestine (Courtesy of Dr. Zhenhong Qu).
2ndPAS-D
Diagnostic Application:
Tumor of glandular origin (e.g., adenocarcinoma) contains polysaccharide glycoprotein
stained pink or red.
Highlights alpha-1-antitrypsin in hepatocytes, a feature of alpha-1-antitrypsin deficiency.
Many fungal microorganisms are also stained by this methods.
Material and Solutions:
FIXATION: Absolute alcohol or 10% formalin - alcohol is preferred. If 10% buffered
neutral formalin is used, there will be a significant loss of glycogen.
TECHNIQUES: Paraffin sections cut at 4 μm.
SOLUTIONS:
Phosphate Buffer Solution pH 6.0*
Sodium chloride ----------------------------------------------------------------- 8.000 gm
Sodium phosphate, dibasic Na HPO -------------------------------------------- 0.282 gm
2 4
Run two slides. Diastase digest one of the slides and then do the PAS stain on both slides.
Staining Results:
Staining attributable to glycogen is selectively eliminated in the diastase digested slide, but will
be stained magenta in the undigested slide.
Lillie, R.D.: Histopathologic Technic and Practical Histochemistry, 3rd Edition, New York,
McGraw-Hill, 1965, pp. 496-497.
Luna, L.G.: Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology,
3rd Edition, New York, McGraw-Hill, 1968, pp. 159, 171.
SOUTHGATE'S MUCICARMINE METHOD
HOME LIST
SOUTHGATE’S MUCICARMINE METHOD
Click the Section Headings (Blue) to Expand/Collapse Material
Image Examples:
Diagnostic Application:
SOLUTIONS:
Weigert’s Iron Hematoxylin (Concentrated)
Solution A
Hematoxylin crystals, C.I. 75290 --------------------------------------------- 2.0 gm
Alcohol, 90% ---------------------------------------------------------------- 100.0 ml
Solution B
Ferric chloride, Fe Cl 6 H 0 62% -------------------------------------------- 4.0 ml
3 2
Heat and boil 3 minutes. Cool and make up to original volume with
50% alcohol. Filter through Pyrex glass wool. This stock solution is
stable for several months when stored in a refrigerator at 3-6 C. o
To prepare the working solution add 30 ml. of distilled water to 10 ml.
of the stock solution.
Staining Procedure:
1. Deparaffinize and hydrate to distilled water.
2. Weigert’s iron hematoxylin for 1 minute.
3. Wash briefly in running water.
4. Decolorize with 0.5% hydrochloric acid in 70% alcohol for 10 seconds.
5. Wash in running tap water for 1 minute and rinse in distilled water.
6. Place in diluted mucicarmine solution for 90 minutes or longer (2-3 hours) if a
more intense stain is desired.
7. Rinse quickly in distilled water.
8. Place in tartrazine solution for 10 seconds.
9. Rinse quickly in distilled water.
10. Dehydrate in graded alcohols.
11. Clear in xylene, three or four changes.
12. Mount with synthetic resin.
Staining Results:
Mucin ---------------------------------------------------------------- deep rose to red
Capsule of Cryptococcus ----------------------------------------- deep rose to red
Nuclei --------------------------------------------------------------- black
Other tissue elements --------------------------------------------- yellow or orange
Comment:
This method gives better staining results than Mayer’s mucicarmine method. It will demonstrate
acidic mucopolysaccharides similar to those stained with Alcian blue (pH 2.5) and with colloidal
iron. For many years we used metanil yellow as the counterstain in this method. This
counterstain has a slight masking effect upon the stained mucin. We found that tartrazine
produces a lighter background stain which allows for better visualization of the mucin.
References:
Culling, C.F.A.: Handbook of Histopathological Technique, 3 ed., Butterworths, London, 1974,
rd
pp. 304-305.
Luna, L.G.: Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology,
3 ed., New York, McGraw-Hill, 1968, pp. 161-162.
rd
MAYER’S MUCICARMINE METHOD
STAINING PROCEDURE:
2. Stain in working Weigert’s Hematoxylin [mix 1 volume of Weigert’s A, (E-320-3), plus 1 volume of
Weigert’s B, (E-320-4)] for approximately seven minutes. Wash in running water for 10 minutes. ***If
using a microwave oven – place slides in working Weigert’s Hematoxylin solution and heat in the
microwave oven for 30 seconds and immediately transfer to tap water.
3. Stain in the working Mucicarmine (mix 1 volume of Mucicarmine Stock, (E-320-1), plus 4 volumes of
tap water) for 60 minutes of longer. Prepare working solution fresh, use once and discard. ***If using a
microwave oven – place slides in Mucicarmine Working Solution and heat in the microwave oven for 60
seconds. The hot stain jar is removed from the oven and allowed to remain at room temperature (22°)
for 5 minutes.
5. Counterstain in Metanil Yellow, 0.25%, (E-320-2) for 15 seconds to 1 minute or longer. (Older
solutions require longer times)
7. Dehydrate in 95% and 100% alcohol’s, two changes; clear through two changes of Xylene.
Nuclei………………………………………………..black
Note: The rose color due to carmin staining will be obscured if sections are overstained with Weigert’s
Hematoxylin and/or Metanil Yellow solution.
REFERENCES: Mallory, F.B.: Pathological Technique, Hafner Publishing Co., N.Y., c. 1961, p. 130
Luna, L.G., (ed), AFIP Manual of Histologic Staining Methods, 3rd. ed., McGraw-Hill Co., NY, c. 1968, pp
161-162.
Sheehan, D.C. & Hrapenak, B.B., Theory and Practice of Histotechnology, 2 nd ed., C.V. Mosby Co., St.
Louis, c. 1980, pp 168-169.
ALCIAN BLUE METHOD (pH 2.5)
STAINING PROCEDURE:
3. Stain in Alcian Blue Solution, (E-323-1) for thirty minutes. Wash in running water for ten minutes and
rinse in distilled water.
4. Counterstain in Nuclear Fast Red Solution, (E-323-2) for five minutes. Wash in running water for one
minute.
5. Dehydrate in 95% Alcohol, Absolute Alcohol, and clear in Xylene, two changes each.
Nuclei stain weakly in formalin-fixed tisssue if Nuclear Fast Red is not used as a counter- stain.
REFERENCES: AFIP Manual of Histological Staining Methods, 3 rd . ed., Ed. L. Luna: New York: McGraw
Hill publications, c. 1968, p. 163.
2. Stain in Alcian Blue Solution, pH 1.0 (E-330-3A) or Alcian Blue, pH 2.5 (E-330-3) for 30 minutes.
3. If Alcian Blue Solution, pH 1.0 is used, blot section dry with filter paper. If Alcian Blue Solution pH 2.5
is used wash in water for 5 minutes.
4. Oxidize in Periodic Acid, 1%, (E-330-1) for 10 minutes. Wash in running water for 5 minutes.
6. Rinse in SodiumMetabisulfite, 0.5%,Aqueous, (E-330-4) three changes, two minutes each. Wash in
running water for ten minutes.
7. Dehydrate in 95% Alcohol, Absolute Alcohol and clearin Xylene, two changes each.
RESULTS:
PAS-Alcian Blue pH 2.5 – All polysaccharides and mucosubstances containing hexoses or deoxyhexoses
with vicinal glycol groups stain magenta to red. Mucosubstances staining red include neutral
mucosubstances. Hyaluronic acid, sialomucins and all but the most strongly acidic sulfated
mucosubstances stain blue.
PAS-Alcian Blue pH 1.0 - PAS positive same as above. Alcinophilic substances at this pH include only the
sulphated mucosaccharides.
REFERENCES: AFIP Manual of Histological Staining Methods, 3rd ed., Ed. L. Luna: New York: McGraw Hill
Publications, c. 1968, p. 168.
Aldehyde groups are formed from carbohydrates (glycogen) within the tissue section via
oxidation after exposure to periodic acid.
The tissue section is then incubated in Schiff reagent, a colorless solution consisting of
basic fuchsin, hydrochloric acid, and sodium metabisulfite.
Chemical bonding between the aldehyde groups and the Schiff reagent results in glycogen
being colored magenta (bright pinkish-red).
The section is finally rinsed with running water, which excites chemical activity releasing
additional chemical groups that intensifies the stain reaction.
The mucicarmine staining procedure is very specific in its detection of mucins of epithelial
origin. It is often used to identify adenocarcinomas, distinguishing these from squamous-cell
carcinomas. It is particularly useful in detecting adenocarcinomas originating from the
gastrointestinal tract.
This procedure can also successfully stain and detect the capsule of a fungal organism known
as Cryptococcus neoformans . This fungus is usually identified in the lungs and in nervous
tissues. C. neoformans is more likely to infect immunocompromised patients than persons with
active immune systems.