Prepared by
ROY ELLIS
IMVS Division of Pathology
The Queen Elizabeth Hospital
Woodville Road, Woodville, South Australia 5011
Principle
The rationale of the technique is that by first staining all the acidic mucins with Alcian blue, those remaining acidic mucins which are also PAS positive will be chemically blocked and will not react further during the technique. Those neutral mucins which are solely PAS positive will subsequently be demonstrated in a contrasting manner. Where mixtures occur, the resultant colour will depend upon the dominant moiety.
The combined result from the port-manteau method demonstrates both acidic- neutral- and mixtures of acidic and neutral mucins.
Method
1. Bring sections to distilled water
2. Stain with Alcian blue for 20 mins
3. Wash well in running tap water for 2 mins
4. Rinse in distilled water
5. Treat with 1% aqueous acetic acid for 6 secs
6. Rinse with distilled water
7. Treat with 0.5% periodic acid for 15 mins
8. Rinse well in distilled water
9. Stain with Schiff's reagent for 20 mins
10. Wash well in running tap water for 5 mins
11. Stain nuclei with alum haematoxylin for 5 mins
12. Wash in running tap water for 2 mins
13. Differentiate with acid alcohol for 1-2 secs
14. Wash and blue nuclei in Scott’s tap water substitute for 30 secs
15. Wash in running tap water for 30 secs
16. Rinse in absolute alcohol
17. Stain with tartrazine in 95% alcohol for 2 mins
18. Dehydrate quickly, clear and mount.
Results
interstitium --------------------------------------- blue
acid mucins --------------------------------------- blue
basement membrane, mesangial matrix ------ magenta
neutral mucins ------------------------------------magenta
erythrocytes -------------------------------------- yellow
Reagent Formulae
1. 1% Alcian blue in 1% acetic acid
Alcian blue 8GX (CI 74240)................... 1 g
Distilled water …............................. 97ml
Glacial acetic acid (Analar).................. 1 ml
Dissolve the Alcian blue in distilled water, heat gently to dissolve. Allow to cool, add the acetic acid.
2. a) Schiff’s reagent (for use with renal biopsies)
Dissolve 3g of Pararosanilin (C.I. 42500) in 600mls of distilled water in a 2 litre conical flask and bring to the boil for 3 minutes. Cool to 50 degrees C and add 30 mls of 1N hydrochloric acid. Cool further to 25 degrees C and add 6 g of sodium metabisulphite ( Univar, M.W. 190.11). Then allow to bleach, in the dark for 24 hours. Add 10g neutral activated decolourising charcoal and shake for 1 minute. Filter rapidly through coarse filter paper (use the fume hood). The solution should be clear or a straw colour. Store in the refrigerator.
Note: Schiffs can be cooled more rapidly by placing the flask in the sink and cooling with running tap water.
b) Pre-prepared Schiff’s reagent - Australian Biostain Pty Ltd,PO Box 1407,Traralgon, VIC.
3. Tartrazine
A saturated solution of tartrazine (CI 19140) in 95% alcohol.
4. 1% Acetic Acid
Acetic Acid (Analar)......................... 1 ml
Distilled water............................... 9 ml
References
Bancroft, J.D. and Stevens, A. 1982Theory and Practice of Histological Techniques. 2nd edition.Churchill-Livingstone.
Cullin, C.F. 1974Handbook of Histopathological and Histochemical Techniques, 3rd edition.Butterworths.