PAS Staining of Fresh Frozen or Paraffin Embedded Human Kidney Tissue
Elizabeth Neumann, Carrie Romer, Jamie Allen, Jeff Spraggins, Maya Brewer, Mark De Caestecker, Angela R.S. Kruse, Jennifer Harvey
Abstract
Scope:
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.
Expected Outcome:
Intermyofibrillar Network…………………………..Pink to Rose
Glycogen………………………………………………….…..Pink to Rose
Myofibrils……………………………………………………..Unstained
Type I fibers………………………………………………….Lighter
Type II fibers…………………………………………………Darker
Blood Vessel walls & connective tissue…..Faintly Stained
Before start
Allow PAS reagents to come to room temperature for ~30 minutes.
Steps
Start with FFPE here:
Allow PAS “kit” to come to room temperature on the bench.
For paraffin sections, deparaffinize in xylene, two changes, 0h 3m 0s each.
Hydrate through graded alcohols, 0h 1m 0s each:
100%, 100%, 95%, 70%, water
Rinse well in distilled water by holding finger over slides, pouring water into sink and adding water. Do this 5 times.
Start Frozen samples here:
Remove frozen slides from freezer and let equilibrate to room temperature, and then place in 10% Formalin for 0h 5m 0s . Proceed to step 7.
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) 0h 3m 0s then in 70% ethanol for 0h 3m 0s . Proceed to step 7.
Rinse with distilled water (follow Step 4).
Place in 0.5% Periodic acid for 0h 10m 0s (to oxidize).
Rinse well in distilled water (follow Step 4).
Pour Schiff’s reagent into Coplin jar containing slides. Allow to sit 15-30 minutes at room temperature (kidney samples ~30 minutes).
Rinse in running warm (~40 °C) distilled or tap water (follow Step 4).
The tissue should appear pink after rinsing with warm water.
Place slides in movable gray slide holder.
Counterstain in Hematoxylin (staining line) for 0h 1m 0s .
Rinse well in distilled water, starting with blue container next to hematoxylin (follow Step 4).
Quickly dip slides into “Bluing” agent (0.5% ammonium hydroxide).
Rinse 1 minute in distilled water until pink color is visible (follow Step 4).
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.
Fix in 2 rounds of xylenes, 0h 1m 0s each (in the hood).
Coverslip slides:
- Place coverslip on paper towel
- Add 2-3 drops of cytoseal to edge (depending on size)
- Dip slide into xylenes, take out and roll the xylene lengthwise on slide
- Line up slide and cover slip and slowly place the slide on the coverslip
- If needed, use dissecting tool to remove bubbles
Image slides using a Leica brightfield scanner, AxioScan Z1 slide scanner (or equivalent) at 20x (~1 µm spatial resolution) and save as .tiff or .jpg file.
