Periodic Acid Schiff (PAS) Reaction
Materials
- Carnoy fixed paraffin
sections
- 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
- Deparaffinize the sections by
passing them through two changes of xylol (2 minutes each).
- Rehydrate the sections by
passing them through 100%, 95%, 70% and 50% alcohols (1 minute each).
- Rinse gently in distilled
water (2 minutes)
- 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)
- Mount in Permount
- 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.