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

[DESCRIPTION]
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. 

[GUIDELINES]
1. Leftover matrix will change the pH of the solutions and prevent adequate staining.

2. Bubbles left on tissue will prevent acquisition of high-quality microscopy images.

[STEPS]
SECTION: Start with FFPE here:
1. Allow PAS “kit” to come to room temperature on the bench.
SECTION: Start with FFPE here:
2. For paraffin sections, deparaffinize in xylene, two changes, 3 min  each.

SECTION: Start with FFPE here:
3. Hydrate through graded alcohols, 1 min  each:
100%, 100%, 95%, 70%, water 

SECTION: Start with FFPE here:
4. Rinse well in distilled water by holding finger over slides, pouring water into sink and adding water.  Do this 5 times.
SECTION: Start Frozen samples here:
5. Remove frozen slides from freezer and let equilibrate to room temperature, and then place in 10% Formalin for 5 min . Proceed to step 7.


SECTION: Start Frozen samples here:
6. 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) 3 min  then in 70% ethanol for  3 min . Proceed to step 7.




SECTION: Start Frozen samples here:
7. Rinse with distilled water (follow Step 4).
SECTION: Start Frozen samples here:
8. Place in 0.5% Periodic acid for 10 min  (to oxidize).


SECTION: Start Frozen samples here:
9. Rinse well in distilled water (follow Step 4).
SECTION: Start Frozen samples here:
10. Pour Schiff’s reagent into Coplin jar containing slides.  Allow to sit 15-30 minutes at room temperature (kidney samples ~30 minutes).


SECTION: Start Frozen samples here:
11. Rinse in running warm (~40 °C) distilled or tap water (follow Step 4).

The tissue should appear pink after rinsing with warm water.
SECTION: Start Frozen samples here:
12. Place slides in movable gray slide holder.
SECTION: Start Frozen samples here:
13. Counterstain in Hematoxylin (staining line) for 1 min .

SECTION: Start Frozen samples here:
14. Rinse well in distilled water, starting with blue container next to hematoxylin (follow Step 4).
SECTION: Start Frozen samples here:
15. Quickly dip slides into “Bluing” agent (0.5% ammonium hydroxide).
SECTION: Start Frozen samples here:
16. Rinse 1 minute in distilled water until pink color is visible (follow Step 4).
SECTION: Start Frozen samples here:
17. 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.
SECTION: Start Frozen samples here:
18. Fix in 2 rounds of xylenes, 1 min  each (in the hood).

SECTION: Start Frozen samples here:
19. 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

SECTION: Start Frozen samples here:
20. 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.