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Alcian Blue and PAS Staining Protocol
NovaUltra Special Stain Kits
This is a combined method utilising the properties of both the PAS (see PAS) and Alcian blue (see Alcian blue) methods to demonstrate the full complement of tissue proteoglycans.
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.
Technical Points
1. Diastase pre-treatment may be used to eliminate the presence of glycogen, prior to staining in Alcian blue.
2. Fixation not-critical, however fixation by aldehyde addition, eg glutaraldehyde, will produce non specific general PAS positivity.
Method
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Bring sections to distilled water.
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Stain with Alcian blue 15 mins
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Wash well in running tap water 2 mins
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Rinse in distilled water
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Treat with periodic acid 5 mins
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Wash well in distilled water
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Stain with Schiff's reagent 10 mins
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Wash well in running tap water 5 mins
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Stain nuclei with haematoxylin 1 min
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Wash in running tap water 2 mins
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Differentiate with acid alcohol
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Wash and blue nuclei in Scott’s tap water
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Wash in water
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Dehydrate, clear and mount
Results
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acidic mucins ............................... blue
- neutral mucins ............................. magenta
- mixtures of above ......................... blue/purple
- nuclei ....................................... deep blue
Reagent Formulae
1. 1% Alcian blue in 3% aq acetic acid (see Alcian blue)
2. 0.5% aq periodic acid
3. Schiff's reagent (see PAS)
References
Mowry RW,(1956),J.Histochem.Cytochem.,4,407