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Renal Alcian Blue/PAS Staining Protocol
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Renal Alcian Blue/PAS Staining Protocol

Prepared by


IMVS Division of Pathology

The Queen Elizabeth Hospital

Woodville Road, Woodville, South Australia 5011


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.


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.


        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


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.

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