Periodic Acid Schiff or PAS Staining: A Method to Assess the Structural Deformities of Glomerulus

JoVE Encyclopedia of Experiments

Kankeronderzoek

0 weergaven • 3:18 min • April 30th, 2023

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The glomerulus is a complex network of capillaries within the kidney that filters waste and removes extra fluid from the blood. The renal filtration rate is dependent on this specialized structure, which can be examined using Periodic Acid Schiff or PAS stain.

To begin, take a paraffinized kidney cortex section on a glass slide. Treat the slide with xylene. Xylene molecules dissolve the paraffin wax from the tissue section. Next, expose the specimen to decreasing concentrations of alcohol. Alcohol removes traces of xylene while rehydrating the tissue section for subsequent binding of the stain.

Now, add periodic acid to the specimen. Periodic acid converts the diols present in the tissue polysaccharides, such as glycogen or mucin, to aldehydes. These aldehydes react with the Schiff's reagent, a fuchsin-sulfurous acid combination, forming a magenta-colored dye that stains the tissue section. Rinse the slide to remove excess stain.

Post rinse, add iron-hematoxylin, a nuclear counterstain that binds to DNA and stains it purple. Wash to remove excess stain. Finally, dehydrate the specimen with increasing strengths of alcohol followed by xylene, which serves as a clearing agent. This step helps improve the contrast of the tissue section for microscopic examination.

Glomerular disease can be visualized as thickening of the basement membrane, altered capillaries, and abnormal cells that relate to impaired renal function.

For detailed visualization of the glomerular cells and mesangial matrix, dry the fixed paraffin-embedded kidney cortex samples at 37 degrees Celsius for 1 hour, followed by deparaffinization with consecutive xylene and descending ethanol immersions. Rehydrate the samples in distilled water for 5 minutes and incubate the slides in Periodic Acid solution in a fume cabinet.

After 5 minutes, rinse the slides with three 5-minute washes in 100 milliliters of distilled water, followed by a 15-minute incubation in Schiff's reagent. At the end of the incubation, wash the slides in running tap water for 5 minutes and counterstain with hematoxylin for 3 seconds before thoroughly rinsing in running tap water for another 15 minutes.

Then, dehydrate the slides with ascending ethanol immersions and two consecutive 3-minute xylene immersions. After air drying, the slides can be mounted with xylene based mounting medium for assessment of their glomerular structure on a light microscope at a 400X magnification.

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Laatst bijgewerkt: 15 augustus 2026