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Malay - Aim of special stain:
- Demonstrate both acid and neutral mucopolysaccharides in tissue samples
- Diagnostic applications:
- Barrett’s oesophagus, mesothelioma, atherosclerosis
- Material supplied for surveys:
- Gastric and colonic tissue composite block cut at 3µm
- Recommended control:
- Samples containing both neutral and acidic mucins – tissues from the gastro-intestinal tract
Alcian Blue PAS method
Reagent – commercial source unknown, unless specified:
| 1. Amylase solution 0.5% | |
| a) α-amylase | 0.5 g |
| b) Distilled water | 100 mL |
| Dissolve the enzyme in the water with gentle shaking. Prepare fresh (can be reused throughout the day). Use either Sigma α-amylase type VI-B from porcine pancreas or α-amylase from Bacillus subtilis (commercial preparation – MP Biomedicals). |
|
| 2. Alcian Blue pH 2.5 – *Note: always add acid to water* | |
| a) Alcian Blue | 1.0 g |
| b) Distilled water | 97 mL |
| c) Glacial acetic acid | 3 mL |
| Dissolve the dye in the distilled water, add acid, mix well. Filter into the reagent bottle and filter before use. Alternatively use commercially prepared reagent (commercial preparation – Point of Care Diagnostics – POCD) – 1% Alcian Blue in 3% Acetic Acid pH 2.5. |
|
| 3. Periodic Acid 1.0% | |
| a) 50% Periodic acid solution | 10 mL |
| b) Distilled water | 500 mL |
| Store refrigerated. | |
| 4. Schiff’s Reagent (commercial preparation – Australian Biostain) | |
| Store refrigerated. Please check the expiry date before use. |
| 5. Haematoxylin |
| 6. Acid alcohol |
| 7. Scott’s tap water |
Method:
*Diastase pre-treatment may be used to eliminate the presence of glycogen, prior to staining in Alcian blue*
Method if Diastase is used with Alcian Blue:
- Bing sections to distilled water.
- Treat sections with amylase solution for 20 minutes.
- Wash well in running tap water for 5 minutes.
- Continue with Alcian Blue PAS.
Alcian Blue staining method:
- Dewax sections and bring to water.
- Stain slides with Alcian blue for 15 minutes.
- Wash well in running tap water for 2 minutes.
- Rinse with distilled water.
- Oxidise in 1% Periodic Acid for 5 minutes.
- Wash in distilled water.
- Treat with Schiff’s reagent for 10 minutes.
- Wash in running tap water for 5 minutes.
- Counterstain with haematoxylin for 1 minute.
- Wash well in running tap water.
- Differentiate with acid alcohol for 3 – 5 seconds.
- Wash in water.
- Blue nuclei in Scott’s tap water.
- Wash in water.
- Dehydrate, clear & mount.
Results:
Acidic mucins – blue
Neutral mucins – magenta
Mixtures of the above – blue/purple
Nuclei – deep blue
* If Diastase was used to remove glycogen, the glycogen will remain unstained in ABPAS + Diastase.*
Disclaimer:
These methods are intended as a guide only. Laboratories that wish to implement these methods should perform internal validation prior to use. The RCPAQAP does not make any claim or warranty for the accuracy or performance of these methods.
References
2020 RCPAQAP Technical General Survey 3.
Russel B Myers, Jerry L Fredenburgh and William E Grizzle. Theory and practice of Histological Techniques. John D Bancroft; Marilyn Gamble. 6th edition. New York, NY: Churchill Livingstone; 2008:172-174.
McManus JFA, Mowry RW. Effects of fixation on carbohydrate histochemistry. Journal of Histochemistry & Cytochemistry. 1958;6(5):309-316.