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Diastase Information and Courses from MediaLab, Inc.

These are the MediaLab courses that cover Diastase and links to relevant pages within the course.

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Histology Special Stains: Carbohydrates
PAS with Diastase: Chemistry

Diastase is often used in conjunction with the PAS staining procedure to specifically identify glycogen granules in tissue samples. Amylase enzymes in a malt extract are used to digest the glycogen, which is then washed out of the tissue sample. It is standard procedure to cut and process two serial sections at the same time. One section is treated with diastase to digest and wash out the glycogen. The other section is left untreated. Both sections are then stained with the PAS procedure for comparative microscopic analysis.

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PAS with Diastase: Staining Protocol

Sample Type Required: Deparaffinized and re-hydrated tissue sections on positively charged slides. Fixative: 10% Neutral Buffered Formalin Step Reagent Time Technical Notes 1 Malt Diastase Solution 1 hour at 37C Solution should be preheated to 37C. Do not heat the solution above 40C as enzyme activity can destroyed at higher temperatures. Insufficient heat may cause incomplete digestion of glycogen. 2 Distilled Water 3 Changes Rinse slides gently to remove diastase. 3 Periodic Acid 0.5% Solution 5 Minutes 4 Distilled Water 3 Changes 5 Schiff Reagent 15 Minutes Gluteraldehyde should be avoided as a tissue fixative since it is a dialdehyde that will react with this reagent and give false-positive PAS staining. 6 Potassium Metabisulfite 0.55% Solution 1 Minute in 2 Changes While some technicians omit this reagent step in the protocol with no problems, metabisulfite rinses may be necessary to remove any excess leucofuchsin left over after exposure to the Schiff reagent. Highly chlorinated water can cause these excess molecules to nonspecifically stain other tissue elements in the section. 7 Running Tap Water Up to 10 Minutes Warm to hot running water develops the PAS stain and gives a more intense staining reaction than cold water. Expected Results Glycogen will be absent from tissue section The following elements will show positive PAS (Rose Red) staining Neutral Mucins Some Epithelial Mucins Basement Membranes Fungal WallsPost Staining Procedure: Tissue sections should be rinsed well in distilled water, dehydrated with 95% and absolute alcohols, cleared and cover-slipped.

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Periodic Acid - Schiff (PAS) with Diastase: Diagnostic Applications

The primary purpose of using the PAS with Diastase staining procedure is to differentiate glycogen from other PAS positive elements such as mucin that may be present in the tissue sample. Mucin can be specifically identified in certain tissue samples using the PAS staining procedure only if the glycogen (which is also PAS-positive) is digested with diastase and washed out. Several enzyme deficiencies can be diagnosed through the analysis of glycogen deposits in the liver. The PAS with Diastase staining procedure can also be used to differentiate glycogen granules from other granules in various tumor types.

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Which special staining technique is most widely used for the demonstration of glycogen in tissue samples?View Page

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