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PERIODIC ACID SCHIFF METHOD
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Small intestine (Courtesy of Dr. Zhenhong Qu) |
FIXATION: 10% buffered neutral formalin, Zenkers or any good fixative.
TECHNIQUE: Paraffin sections cut at 4 μm.
SOLUTIONS:
0.5% Periodic Acid Solution
Periodic acid --------------------------------------------------------------------- 0.5 gm
Distilled water ------------------------------------------------------------------- 100.0 ml
Modified Lillies Schiff Solution
Dissolve 0.3 gm of pararosanilin, in 20 ml of 20% ethyl alcohol. Add 80 ml
of distilled water, 2.0 gm of sodium metabisulfite, 0.15 gm of sodium hydrosulfite,
and thoroughly mix. Then add 1.0 ml of hydrochloric acid and 0.3 gm of activated charcoal. Stir for 3 minutes and filter through Whatman #1 filter paper. The solution should be water-white. Store Schiffs reagent in a refrigerator at 3-6° C. When stored in a refrigerator, the solution has a shelf-life of at least one year.
0.3% Sodium Borate
Sodium borate (Na2 B4 O7 10H2O) ------------------------------------------- 0.3 gm
Distilled water ---------------------------------------------------------------- 100.0 ml
1 . Deparaffinize and hydrate to distilled water.
2. Oxidize in periodic acid solution for 10 minutes. For kidney, skin and diastase digested liver biopsies, 15 minutes.
3. Rinse in four changes of distilled water.
4. Place in modified Lillies Schiff solution for 15 minutes. For kidney and skin biopsies, 30 minutes.
5. Rinse in four changes of distilled water.
6. Place in 0.3% sodium borate for 15 seconds.
7. Rinse in four changes of distilled water.
8. Stain in acidified Lillie-Mayer hematoxylin for 45 seconds.
11. Rinse in four changes of distilled water.
12. Dehydrate through graded alcohols.
13. Clear in three or four changes of xylene.
14. Mount with synthetic resin.
Glycogen, mucin, reticulum, colloid droplets, hyaline deposits in glomeruli, most basement membranes, colloid of pituitary stalks and thyroid give a rose to purple reaction; some types of fungi - red, nuclei - blue.
Treatment of tissue sections with 0.5% periodic acid results in the formation of aldehyde groupings by cleavage of 1,2 glycols found in various carbohydrates. The presence of the newly formed aldehyde groups is detected by Schiff’s reagent (or leucofuchsin). The first stage reaction involves the formation of a colorless, unstable dialdebyde addition compound that is transformed to the colored final product by restoration of the quinoid chromophoric grouping of pararosanilin or other basic fuchsin. This color restoration is usually accomplished by washing in running tap water for 10 minutes after treatment with Schiff’s reagent. We found that treatment with 0.3% sodium borate for 15 seconds accomplishes the same purpose.
The formula for Lillies Schiff reagent has been modified. This modified solution requires less basic fuchsin (pararosanilin) to prepare, can be prepared much faster, yields equally good staining results, and appears to have greater stability than Lillies formula.
Schiff’s reagent after decolorization with activated charcoal may be either water clear or have a pale straw color. Some believe that the quality of the activated charcoal determines the final color of the Schiff’s reagent. Fresh, activated charcoal is supposed to result in a water clear Schiff’s reagent. From our experience this is not necessarily true as we have found that Schiff’s reagent prepared with different samples of basic fuchsin will not all decolorize completely even when fresh activated charcoal is used. The final color of the Schiff’s reagent does not appear to affect the quality or intensity of the Schiff’s reaction.
Schiff’s reagent can be tested by pouring a few drops of the reagent into 10 ml of reagent grade formaldehyde (37-40%) in a small beaker. If the solution turns reddish purple rapidly, it is good. If the reaction is delayed and the resulting color deep blue-purple, the solution is breaking down and should be discarded.
Garvey, W. et al: Combined modified periodic acid-Schiff and batch staining method. J. Histotechnol. 15:117-120, 1992.
McManus, J.F.A. and Mowry, R.W.: Staining Methods Histologic and Histochemical, New York, Paul B. Hoeber, 1960, pp. 126-128.
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