TITLE: PAS Staining of Fresh Frozen or Paraffin Embedded Human Kidney Tissue
AUTHORS: Elizabeth Neumann, Jamie Allen, Jennifer Harvey, Maya Brewer, Carrie  Romer, Mark De Caestecker, Jeff Spraggins

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Scope:</div><div class = "text-block">The PAS stain is used to demonstrate polysaccharides such as glycogen, and mucosubstances such as glycoproteins, glycolipids and mucins in tissues.  It is used as a replacement for the H&E in kidney pathology.</div><div class = "text-block">Expected Outcome:</div><div class = "text-block">Intermyofibrillar Network…………………………..Pink to Rose</div><div class = "text-block">Glycogen………………………………………………….…..Pink to Rose</div><div class = "text-block">Myofibrils……………………………………………………..Unstained</div><div class = "text-block">Type I fibers………………………………………………….Lighter</div><div class = "text-block">Type II fibers…………………………………………………Darker</div><div class = "text-block">Blood Vessel walls & connective tissue…..Faintly Stained</div></div>

[STEPS]
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Allow PAS “kit” to come to room temperature on the bench.
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For paraffin sections, deparaffinize in xylene, two changes,   each.
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Hydrate through graded alcohols,   each:100%, 100%, 95%, 70%, water
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Remove frozen slides from freezer and let equilibrate to room temperature, and then place in 10% Formalin for  . Proceed to step 7.
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If staining is performed following MALDI analysis and samples have matrix on them, remove matrix in 90% ethanol (~2-3 min or until matrix is gone)   then in 70% ethanol for   . Proceed to step 7.
[Until Matrix is removed]
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Rinse with distilled water (follow Step 4).
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Place in 0.5% Periodic acid for   (to oxidize).
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Rinse well in distilled water (follow Step 4).
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Pour Schiff’s reagent into Coplin jar containing slides.  Allow to sit 15-30 minutes at room temperature (kidney samples ~30 minutes).
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Rinse in running warm (~40 °C) distilled or tap water (follow Step 4).The tissue should appear pink after rinsing with warm water.
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Place slides in movable gray slide holder.
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Counterstain in Hematoxylin (staining line) for  .
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Rinse well in distilled water, starting with blue container next to hematoxylin (follow Step 4).
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Quickly dip slides into “Bluing” agent (0.5% ammonium hydroxide).
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Rinse 1 minute in distilled water until pink color is visible (follow Step 4).
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Dehydrate through graded alcohols, 10 short dips in each:95%, 95%, 100%, 100%If the last 100% ethanol rinse is colored by residual stain, additional washing in new 100% ethanol solution should be performed.
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Fix in 2 rounds of xylenes,   each (in the hood).
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Coverslip slides:Place coverslip on paper towelAdd 2-3 drops of cytoseal to edge (depending on size)Dip slide into xylenes, take out and roll the xylene lengthwise on slideLine up slide and cover slip and slowly place the slide on the coverslipIf needed, use dissecting tool to remove bubbles
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Rinse well in distilled water by holding finger over slides, pouring water into sink and adding water.  Do this 5 times.
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Image slides using a Leica brightfield scanner (or equivalent) at 20x (~1 µm spatial resolution) and save as .tiff or .jpg file.