1. Periodic Acid Schiff (PAS) Stain 
NOTE: The PAS stain will highlight the basement membranes of glomerular capillary loops and the tubular epithelium. It enables detailed visualization of the glomerular cells, mesangial matrix and potential expansion, and potential changes of the GBM (i.e., thickening and irregularities).
<ol>
	<li>Section the paraffin-embedded, PFA-fixed kidney cortex using a microtome at 5 &#181;m thickness onto poly-L-lysine coated slides. Dry at 37 &#176;C for 1 h. Ensure the section does not contain any folds or holes, which can distort the morphology under the microscope.</li>
	<li>Deparaffinize slides by incubating twice in xylene (CAUTION, irritant; use in fume cabinet) for 3 min each, twice in 100% EtOH for 3 min each, and then once in 95%, 70%, and 50% EtOH for 3 min each, all at room temperature. Rehydrate the slides in dH2O.</li>
	<li>Incubate the slides in periodic acid solution (CAUTION, irritant; use in fume cabinet) (1 g/dL) for 5 min, and then rinse the slides in several changes of dH2O. Use a container with 100 mL dH2O at room temperature.</li>
	<li>Incubate slides in Schiff's reagent (Parasoaniline HCl 6 g/L and sodium metabisulfite 4% in HCl 0.25 mol/L) for 15 min at room temperature. Wash slides in running tap water for 5 min.</li>
	<li>Counterstain with Hematoxylin for 3 s before thoroughly rinsing slides in running tap water for 15 min. 
	NOTE: Some optimization may be required to determine the optimal time for Hematoxylin staining.</li>
	<li>Dehydrate slides using the reverse of the deparaffinization protocol. Finish with xylene.</li>
	<li>Air dry slides and mount with xylene-based mounting media.</li>
	<li>Image on a light microscope at 400X&#160;magnification to assess glomerular structures. Evaluate the following: thickening and irregularities of the GBM, collapsing of capillary loops, fibrotic tissue, sclerosis, cellular proliferation (endothelial, podocyte, and mesangial, or inflammatory cells infiltrating the tuft). 
	NOTE: For a comprehensive evaluation of glomerular pathophysiology, lesions elsewhere in the kidney should be evaluated, such as in the tubules.</li>
</ol>

<table><tbody><tr><td>Periodic Acid-Schiff (PAS) Staining System</td><td>&amp;nbsp;Sigma-Aldrich</td><td>&amp;nbsp;395B-1KT</td><td></td></tr><tr><td>Hematoxylin</td><td>&amp;nbsp;Sigma-Aldrich</td><td>&amp;nbsp;H3136</td><td></td></tr><tr><td>Xylene</td><td>&amp;nbsp;MerckMillipore</td><td>&amp;nbsp;108298</td><td></td></tr><tr><td>Poly-Prep Slides</td><td>&amp;nbsp;Sigma-Aldrich</td><td>&amp;nbsp;P0425-72EA</td><td></td></tr><tr><td>Mounting Medium</td><td>&amp;nbsp;ThermoFisher Scientific</td><td>&amp;nbsp;8030</td><td></td></tr><tr><td>Hydrophobic Pen</td><td>&amp;nbsp;Abcam</td><td>&amp;nbsp;ab2601</td><td></td></tr><tr><td>Fluorescent Light Miscroscope</td><td>&amp;nbsp;Leica Microsystems</td><td></td><td></td></tr><tr><td>Image J Analysis Software</td><td>&amp;nbsp;Image J</td><td></td><td></td></tr></tbody></table>

periodic acid schiff or pas staining: a method to assess the structural deformities of glomerulus

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

Source: Stevens, M., Oltean, S. <a href='https://www.jove.com/video/57764' target='_blank'>Assessment of Kidney Function in Mouse Models of Glomerular Disease.</a>&#160;J. Vis. Exp. (2018)
In this video, we describe the protocol for performing Periodic Acid Schiff (PAS) stain to assess the structural anomalies in the kidney glomerulus. The PAS staining method enables appropriate evaluation of the glomerulus pathophysiology.

Source: Stevens, M., Oltean, S. Assessment of Kidney Function in Mouse Models of Glomerular Disease.&#160;J. Vis. Exp. (2018)
In this video, we describe ...

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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7.1K Views. 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 alco...

Abstract

Protocol

Disclosures

<table><tbody><tr><td>Periodic Acid-Schiff (PAS) Staining System</td><td>&nbsp;Sigma-Aldrich</td><td>&nbsp;395B-1KT</td><td></td></tr><tr><td>Hematoxylin</td><td>&nbsp;Sigma-Aldrich</td><td>&nbsp;H3136</td><td></td></tr><tr><td>Xylene</td><td>&nbsp;MerckMillipore</td><td>&nbsp;108298</td><td></td></tr><tr><td>Poly-Prep Slides</td><td>&nbsp;Sigma-Aldrich</td><td>&nbsp;P0425-72EA</td><td></td></tr><tr><td>Mounting Medium</td><td>&nbsp;ThermoFisher Scientific</td><td>&nbsp;8030</td><td></td></tr><tr><td>Hydrophobic Pen</td><td>&nbsp;Abcam</td><td>&nbsp;ab2601</td><td></td></tr><tr><td>Fluorescent Light Miscroscope</td><td>&nbsp;Leica Microsystems</td><td></td><td></td></tr><tr><td>Image J Analysis Software</td><td>&nbsp;Image J</td><td></td><td></td></tr></tbody></table>

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

In This Article

Abstract

Protocol

Disclosures

Materials

Tags

Name	Company	Catalog Number
Periodic Acid-Schiff (PAS) Staining System	Sigma-Aldrich	395B-1KT
Hematoxylin	Sigma-Aldrich	H3136
Xylene	MerckMillipore	108298
Poly-Prep Slides	Sigma-Aldrich	P0425-72EA
Mounting Medium	ThermoFisher Scientific	8030
Hydrophobic Pen	Abcam	ab2601
Fluorescent Light Miscroscope	Leica Microsystems
Image J Analysis Software	Image J