Pas, Alcian blue and massons trichrome stains.

PERIODIC ACID SCHIFF REACTION (PAS)
Purpose: It is required to demonstrate
carbohydrates e.g., neutral muco-
polysaccharides, mucoproteins and
glycoproteins in the tissue. It can also be used to
demonstrate fungi.
Principle: Aldehyde is generated by oxidation of
1:2 glycol groups present in carbohydrates by
periodic acid. This combines with Schiff’s
reagent to form a coloured compound in situ.
Requirements
1. 0.1% periodic acid
2. 0.5% sodium metabisulphite
3. Schiff’s solution (commercially available)
4. Haematoxylin as in H&E stain
Procedure
1. Rehydrate as for H&E staining.
2. Rinse in tap water for 5 min.
3. Rinse in distilled water by giving 15 dips.
4. Oxidise in 0.1% periodic acid for 15 min.
5. Wash well in tap water for 5 min.
6. Rinse well in 3 changes of distilled water
giving 5 dips in each.
7. Treat with Schiff’s reagent for 10 min.
8. Treat with 3 changes of 0.5% sodium
metabisulphite for 2 min each.

9. Wash in tap water.
10. Stain in hematoxylin as desired.
11. Dehydrate, clear and mount as for H&E
stain.
Result: Neutral mucopolysaccharides,
mucoproteins and glycoproteins stain pink to
magenta whereas nuclei stain blue. Fungi also
stain magenta.
Alcian blue stain:

Purpose: It is used for distinguishing between
Mucin-secreting adenocarcinoma and
Undifferentiated squamous cell carcinoma, and to identify naturally occurring

carbohydrates in Tissue.
Principle: Connective tissue ground substance
Is coloured only with Alcian blue because it lacks
Sufficient polysaccharides to react with PAS.
Epithelial mucin or glycogen will react with PAS
And not at all with Alcian blue. Complex
Carbohydrates, such as epithelial mucin
Secretions, stain with both Alcian blue and PAS.
Control: Small intestine
Requirements
1. Alcian blue prepared by dissolving 01 g
Alcian blue in 100 ml of 3% glacial acetic
Acid (pH should be 2.5).
2. 3% Glacial acetic acid prepared by diluting 3
Ml glacial acetic acid in 97 ml of distilled Water.
3. Nuclear Fast red counter stain prepared by
Dissolving 0.1g nuclear fast red, 5 g
Aluminium sulphate and one crystal of
Thymol in 100 ml distilled water. Heat water
To 60°C and then add to it aluminium
Sulphate and stir until dissolved. Then add
Nuclear fast red and cool to 50°C. Filter and
Add thymol crystal. Store in refrigerator.
4. 1% Periodic acid
5. Schiff’s reagent
Procedure for Alcian Blue stain

1. Rehydrate the section.
3. Stain in Alcian blue for 20 min.
5. Place in nuclear fast red counter stain for 3-5 min.
6. Rinse in tap water for 15 dips.
7. Dehydrate, clear and mount.
Results: Acid mucopolysaccharides stain blue
Whereas other tissue elements stain red.
MASSON'S TRICHROME STAIN

Purpose: To differentiate connective tissue
elements.
Fixative: The tissues should be fixed in Bouin's
or Zenker’s fluid. Formalin fixed sections should
be mordanted in Zenker's fluid overnight at room
temperature or at 56°C for one hour.
Requirements
1. Weigert's iron haematoxylin
a. Solution A
i) Iron hematoxylin 1.0 g
ii) 95% alcohol 100 ml
b. Solution B
i) 29% aqueous Ferric Chloride 4 ml
ii) Distilled water 95 ml
iii) Concentrated HCl 1 ml
c. Working solution: Mix equal parts of
solutions A and B
2. Biebrich scarlet acid fuchsin solution:

Prepared by mixing 1 ml glacial acetic acid,
10 ml 1% aqueous acid fuchsin in 90 ml of
1% aqueous Biebrich scarlet.
3. Phosphomolybdic-Phosphotungstic acid
solution. Prepared by dissolving 2.5 g each
of phosphomolybdic acid and
phosphotungstic acid in 100 ml distilled
water.
4. Aniline blue solution prepared by dissolving
2.5 g aniline blue in 2 ml acetic acid and 100
ml distilled water.
Procedure:
1. Bring sections to water as usual.
2. Rinse in distilled water.
3. Stain in Weigert's iron haematoxylin for 10
min.
4. Wash in running tap water for 10 min.
6. Stain in Biebrich scarlet-acid fuchsin solution
for 5 min.
8. Place in aqueous phosphomolybdic acid-
phosphotungstic acid solution for 10 min.
9. Drain slide, and pour on it aniline blue
solution for 5 min.
11. Differentiate in 1% acetic acid for 3 min.

12. Dehydrate in absolute alcohol clear in
xylene and mount in Canada balsam or
DPX.
Results: Nuclei stain blue black whereas
cytoplasm, muscle and keratin granules stain
red. Collagen, cartilage, mucin and basophil
granules are stained blue.

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PERIODIC ACID SCHIFF REACTION (PAS)

Purpose: It is required to demonstrate

carbohydrates e.g., neutral muco-

polysaccharides, mucoproteins and

glycoproteins in the tissue. It can also be used to

Principle: Aldehyde is generated by oxidation of

1:2 glycol groups present in carbohydrates by

periodic acid. This combines with Schiff’s

reagent to form a coloured compound in situ.

1. 0.1% periodic acid

2. 0.5% sodium metabisulphite

3. Schiff’s solution (commercially available)

4. Haematoxylin as in H&E stain

1. Rehydrate as for H&E staining.

2. Rinse in tap water for 5 min.

3. Rinse in distilled water by giving 15 dips.

4. Oxidise in 0.1% periodic acid for 15 min.

5. Wash well in tap water for 5 min.

6. Rinse well in 3 changes of distilled water

giving 5 dips in each.

7. Treat with Schiff’s reagent for 10 min.

8. Treat with 3 changes of 0.5% sodium

metabisulphite for 2 min each.

10. Stain in hematoxylin as desired.

11. Dehydrate, clear and mount as for H&E

Result: Neutral mucopolysaccharides,

mucoproteins and glycoproteins stain pink to

magenta whereas nuclei stain blue. Fungi also

Alcian blue stain:

Mucin-secreting adenocarcinoma and

Undifferentiated squamous cell carcinoma, and to identify naturally occurring

Is coloured only with Alcian blue because it lacks

Sufficient polysaccharides to react with PAS.

Epithelial mucin or glycogen will react with PAS

And not at all with Alcian blue. Complex

Carbohydrates, such as epithelial mucin

Secretions, stain with both Alcian blue and PAS.

Control: Small intestine

1. Alcian blue prepared by dissolving 01 g

Alcian blue in 100 ml of 3% glacial acetic

Acid (pH should be 2.5).

2. 3% Glacial acetic acid prepared by diluting 3

Ml glacial acetic acid in 97 ml of distilled Water.

3. Nuclear Fast red counter stain prepared by

Dissolving 0.1g nuclear fast red, 5 g

Aluminium sulphate and one crystal of

Thymol in 100 ml distilled water. Heat water

To 60°C and then add to it aluminium

Sulphate and stir until dissolved. Then add

Nuclear fast red and cool to 50°C. Filter and

Add thymol crystal. Store in refrigerator.

Procedure for Alcian Blue stain

Results: Acid mucopolysaccharides stain blue

Whereas other tissue elements stain red.

MASSON'S TRICHROME STAIN

Fixative: The tissues should be fixed in Bouin's

or Zenker’s fluid. Formalin fixed sections should

be mordanted in Zenker's fluid overnight at room

temperature or at 56°C for one hour.

1. Weigert's iron haematoxylin