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Alcian Blue Staining Protocol
Description:
Alcian
blue stains acid mucosubstances and acetic mucins. Excessive amounts
of non-sulfated acidic mucosubstances are seen in mesotheliomas,
certain amounts occur normally in blood vessel walls but increase in
early lesions of atherosclerosis. Strongly acidic mucosubstances
will be stained blue, nuclei will be stained pink to red, and
cytoplasm will be stained pale pink.
Fixation:
formalin
fixed, paraffin embedded tissue sections.
Solutions and Reagents:
3% Acetic Acid
Solution:
Glacial acetic acid ----------------- 3 ml
Distilled water --------------------- 97 ml
Alcian Blue Solution
(pH 2.5):
Alcian blue, 8GX -------------------- 1 g
Acetic acid, 3% solution ----------- 100 ml
Mix well and adjust pH to 2.5 using acetic acid.
Nuclear fast red ------------------- 0.1 g
Aluminum
sulfate------------------ 5 g
Distilled water ---------------------100 ml
Dissolve aluminum sulfate in water. Add nuclear fast red and slowly heat to boil and cool. Filter and add a grain of thymol as a preservative.
Procedure:
1.
Deparaffinize slides and
hydrate to distilled water.
2.
Stain in
alcian blue solution for 30 minutes.
3.
Wash in running tap water
for 2 minutes.
4.
Rinse in distilled water.
5.
Counterstain in nuclear
fast red solution for 5 minutes.
6.
Wash in running tap water
for 1 minute.
7.
Dehydrate
and through 95% alcohol, 2 changes of absolute alcohol, 3 minutes
each.
8.
Clear in
xylene or xylene substitute.
9.
Mount with resinous
mounting medium.
Results:
Strongly acidic sulfated mucosubstances -------------- blue
Nuclei ------------------------------------------------------
pink to red
Cytoplasm -------------------------------------------------
pale pink
Positive Controls:
Small intestine, appendix, or colon.