Exercise 2.3 - Periodic Acid Schiff (PAS) Reaction

LEVEL I

Materials

• Carnoy fixed paraffin sections 1
• Ethanol series, 50, 70, 95 and 100% (v/v)
• 1% Amylase in 0.1 M sodium phosphate buffer, pH 7.0
• 0.1 M Sodium phosphate Buffer, pH 7.0
• Incubator at 37° C
• Periodic acid
• Schiff's Reagent
• Sulfurous acid
• Xylol, Permount and coverslips
• Microscope

Procedure

1. Deparaffinize the sections by passing them through two changes of xylol (2 minutes each).

2. Rehydrate the sections by passing them through 100%, 95%, 70% and 50% alcohols (1 minute each).

3. Rinse gently in distilled water (2 minutes)

4. Pass the slides sequentilly through the following:
   • Place a slide in 1% buffered amylase at 37° C (1 hour). Place another slide in buffer at 37° C (1 hour)
   • Oxidize both slides in periodate (5 minutes)
   • Rinse gently in fresh distilled water (1 minute)
   • Stain in Schiff's reagent (15--30 minutes)
   • Bleach in sulfurous acid to remove any non--specific stain (15-30 minutes)
   • Rinse gently in fresh distilled water (5 minutes)
   • 70% alcohol (5 minutes)
   • 95% alcohol (5 minutes)
   • 100% alcohol (5 minutes)
   • Xylol (5 minutes)

5. Mount in Permount

6. Compare the amylase treated slides with those not so treated. Amylase removes amylose, animal starch. Those structures present without enzyme treatment, but missing after treatment are composed of amylose.
   Remember that the staining reagent is for aldehydes. Any compound with an aldehyde will turn pink with this reaction. Careful controls must always be used and even greater care taken in the final analysis of stained structures.

Notes

The major carbohydrates which remain in cells affixed to slides are the rather insoluble glucose polymers: glycogen, starch, and cellulose. All three polysaccharides give a positive periodic acid--Schiff (PAS) reaction. This staining procedure can be used on a variety of plant and animal material to determine the presence and intracellular localization of these polysacchrides.

Periodic acid is an oxidizing agent which breaks the C- -C bond between two adjacent hydroxyl groups. The 1,2--diol group in glucose is converted into a dialdehyde and any carbonyl groups are converted to carboxilic acids. The advantage of periodic acid lies in the specificity of its oxidation. It forms aldehydes within the polysacchride molecule but it does not continue the oxidation of the polymers to low molecular weight water soluble forms. Thus, glycogen, starch, and cellulose contain dialdehyde groups after the periodate treatment and are left in the cell in insoluble forms which can then be treated with Schiff's aldehyde reagent to form a purple colored product.

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