Periodic acid is an oxidizer. The reaction of periodic acid oxidizes the vicinal diols in some sugars such as glycogen, glycoproteins, glycolipids and mucins, usually breaking up the bond between adjacent carbons and creating a pair of aldehydes at the two free tips of each broken monosaccharide ring. The exposed aldehydes can be stained by magenta and generate red to purple red complex, which is a position mark.
1. Deparaffinize and rehydrate by immersing the slides through the following:
1.1. Xylene: wash twice, 20 minutes each time
1.2. 100% alcohol: wash twice, 5 minutes each time
1.3. 75% alcohol: wash for 5 minutes
1.4. Wash briefly in tap water
2. Periodic acid stain
Immerse slides in periodic acid solution for 15 min, wash in tap water and twice in distilled water.
3. Schiff stain
Stain slides in Schiff reagent for 30 min and keep in dark, followed by washing in running tap water for 5 min.
4. Hematoxylin stain
Rinse slides in hematoxylin solution for 1-3 min, wash in tap water. Differentiate in 1% hydrochloric acid for a few second, wash in tap water. Bluing in 0.2% ammonia water, then wash in tap water.
5. Dehydrate and sealing:
Dehydrate in absolute alcohol for 3 times, 5 min each time clear in xylene for 2 times, 5 min each time, seal with neutral gum.
6. Microscopy detection and analysis.
Purple red for collagen, goblet cell, fungus, plant starch granule and cell wall. Blue for nuclei.
1. The periodic acid solution should be stored in 4℃.
2. The Schiff reagent should be stored at 4°C, avoiding light and reach room temperature before usage. The Schiff reagent can be recycled and reused for many times. When it turns pink, it is unusable.
3. The staining time of Schiff reagent depends on room temperature. Shorten the staining time when room temperature is high (e.g. in summer), and extend it to about 60 min when room temperature is low (e.g. in winter).