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Author: Kelsey Goldman, HTL (ASCP)

Last Update: 22 March 2025

Copyright: 2025, HistologyOutlines.com

Cite this Page! -> Goldman, Kelsey. “Periodic Acid- Schiff.” HistologyOutlines.Com, 22 March 2025. Accessed (today).

Purpose

Periodic Acid Schiff (PAS) is a highly versatile stain that has a wide variety of applications. The PAS is used for the detection of polysaccharides, neutral mucocutaneus and basement membranes. A diastase digestion can be added to remove glycogen to give better visualization what staining belongs to the glycogen and what belongs to other elements in the tissue (Carson, 2001) (Suvarna et al., 2019).
PAS can be used to demonstrate increased thickness of the basement membrane of the kidney which is an indicator of a number of conditions (Suvarna et al., 2019). PAS also can be used in demonstrating the presence of fungus in tissue as the cell wall of fungi contain a number of polysaccharides that will react with periodic acid ( Suvarna et al., 2019).

Control Tissues

The PAS and all of the subcategories of the stain rely on the same basic set of chemical reactions:

Aldehydes in the tissue are oxidized by the periodic acid (most commonly 1,2 glycol groups found on proteins) (Carson, 2001).
Schiff’s reagent is applied, and this creates the Schiff reaction. This is a reduction that creates a colorless compound called leucofuchsin (Carson, 2001).
The chemically reduced slides are washed in running tap water. This leads to the loss of a sulfurous group on the central carbon of what was the aldehyde. The loss of the sulfur leads to the classic pink color (Carson, 2001).
Metabisulfite removes any excess Schiff on the slide so that it cannot continue to react (Carson, 2001).

Carbohydrates are molecular rings present in many biological compounds. Most notably, they link together to form polysaccharides (or sugar chains) and can be used as fuel. Many of them contain a side reactive group called an aldehyde. This aldehyde group is oxidized by periodic acid. This results in a broken ring that can now react with Schiff’s reagent. Schiff’s reagent binds to the broken carbon ring. This binding leads to a colorless compound. However, once running tap water is introduced to the tissue, the sulfonate is dislocated off the central atom, and a brilliant rose/red color can be seen (Suvarna et al., 2019).
Periodic acid as an oxidizer strikes the perfect balance that gives this stain its essence. It is effective as an oxidizing agent but not so effective that it removes the aldehydes altogether (Davenport, 1969).
Schiff’s reagent is what accomplishes the detection of the aldehyde. Schiff contains basic fuchsin and sulfurous acid (Davenport, 1969).

Use 10% NBF or Bioun’s as a fixative. Use methyl alcohol for 10-15 minutes on blood smears that are intended to stain with PAS (Carson, 2001).
Do not use glutaraldehyde-based fixatives. The glutaraldehyde contains two aldehyde groups. If there is excess or not fully bound reagent, then it will react with the PAS reagents (Carson, 2001).
Washing in tap water after the sodium metabisulfite step is critical for proper color development (Carson, 2001).
Sections stained with a diastase digestion with malt diastase are more likely to fall off the slide (Carson, 2001).
Some histology labs use the very old practice of forgoing a synthetic malt diastase digestion. They simply spit onto the slide, and the amylase in the saliva breaks down the glycogen (Carson, 2001). Please do not do this. Please embrace modernity.
If performing a diastase digestion, diastase will not be able to appropriately digest slides fixed in picric acid (Carson, 2001).
Leaving sections for too long in periodic acid will over-oxidize the tissue. This can lead to excessive and nonspecific staining (Disbrey, 1970).

Carson, F. L. (2001). Histotechnology: a self-instructional text (2nd ed., [Reprint]). ASCP Press.
Davenport, H. (1969). Histological and Histochemical Technics. W B Saunders Company.
Disbrey, B. (1970). Histological Laboratory Methods. E & S Livingstone.
Suvarna, K. S., Layton, C., & Bancroft, J. D. (2019). Bancroft’s theory and practice of histological techniques (8th ed.). Elsevier.

Deparaffinize and rehydrate slides.
Place slide in a diastase solution that has been preheated to 37°C. Many labs use a container within a water bath. Leave slides in the solution for 1 hour. Do not place the "without diastase” control in this solution. Hold this slide in deionized water for 1 hour.
Place slides into the 0.5% periodic acid for 5 minutes.
Prepare three changes of fresh distilled water and pass slides through each container.
Warm Schiff’s reagent to room temperature by leaving the reagent in the counter. Stain slides in Schiff reagent for 15 minutes.
Pass slides through two changes of 0.5% sodium metabisulfite.
Wash slides in running tap water for 10 minutes.
Counterstain for 30 seconds in working Harris hematoxylin (48 mL Harris hematoxylin with 2 mL acetic acid).
Wash sections in running water to blue the Harris hematoxylin.
Dehydrate to xylene and coverslip with a xylene-based mounting media.

Deparaffinize and rehydrate slides.
Place slide in a diastase solution that has been preheated to 37°C. Many labs use a container within a water bath. Leave slides in the solution for 1 hour. Do not place the "without diastase” control in this solution. Hold this slide in deionized water for 1 hour.
Place slides into the 0.5% periodic acid for 5 minutes.
Prepare three changes of fresh distilled water and pass slides through each container.
Warm Schiff’s reagent to room temperature by leaving the reagent in the counter. Stain slides in Schiff’s reagent for 15 minutes.
Pass slides through two changes of 0.5% sodium metabisulfite.
Wash slides in running tap water for 10 minutes.
Counterstain for 30 seconds in working Harris hematoxylin (48 mL Harris hematoxylin with 2 mL acetic acid).
Wash sections in running water to blue the Harris hematoxylin.
Dehydrate to xylene and coverslip with a xylene-based mounting media.