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Alcian Blue Staining Protocol
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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.0.1% Nuclear Fast Red Solution:

      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.


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.


      Strongly acidic sulfated mucosubstances -------------- blue

      Nuclei ------------------------------------------------------ pink to red

      Cytoplasm ------------------------------------------------- pale pink 

Positive Controls:

Small intestine, appendix, or colon.

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