TITLE: Periodic acid Schiff hematoxylin (PASH) staining for human retina
AUTHORS: Dongfeng Cao, Jeff D. Messinger, Angela  Kruse, Jamie Allen, David       Anderson, Christine A. Curcio, Jeff Spraggins

[DESCRIPTION]
Periodic acid Schiff hematoxylin (PASH) staining protocol is adapted from other tissues to identify carbohydrate in human retinal sections in our lab. This method is used for labeling 10-12 µm thick cryosections of human retina, RPE, choroid, and sclera, preserved within 6 hours of death in 4% paraformaldehyde in 0.1 M phosphate buffer, picked up on SuperFrost glass slides, and stored at -80°C. It is intended to be counterstained for nucleic acids in nuclei using modified Harris hematoxylin. 

The most common application is for demonstrating glycogen in liver tissue. Positive staining for glycogen is magenta, and nuclei stained by hematoxylin are blue. 

This PASH staining protocol is helpful to identify drusen, basal laminar deposits, retinal pigment epithelium (RPE, containing abundant lipofuscin), nuclei, and cell layers of the retina, choroid, and sclera. It is preferable to traditional H&E staining for structures and pathology specific to age related macular degeneration (AMD). It is particularly useful for diagnosis and comparison to clinical retina imaging, especially optical coherence tomography. Since the late 1960s, PASH applied to human AMD specimens was noted to stain an eosinophilic material at the base of the RPE (now known as basal laminar deposit), drusen (the characteristic lesions), and Bruch’s membrane. [1-4]

1.Gass JDM. Pathogenesis of disciform detachment of the neuroepithelium. III. Senile disciform macular degeneration. Am. J. Ophthalmol. 1967;63:617-644. PMID 6019308 
2.Farkas TG, Sylvester V, Archer D, Altona M. The histochemistry of drusen. American Journal of Ophthalmology. 1971;71(6):1206-1215. PMID 4253686 
3.Sarks SH. Ageing and degeneration in the macular region: a clinico-pathological study. Br. J. Ophthalmol. 1976;60(5):324-341. PMID 952802 
4.Vogt SD, Curcio CA, Wang L, Li C-M, McGwin G, Jr, Medeiros NE, Philp NJ, Kimble JA, Read RW. Retinal pigment epithelial expression of complement regulator CD46 is altered early in the course of geographic atrophy. Exp Eye Res. 2011;93( 4):413-423 PMID 21684273 

[STEPS]
SECTION: PASH Staining
1. Remove glass slides with human retinal cryosections from -80 °C, keep on ice during transfer from freezer to bench.

SECTION: PASH Staining
2. Put glass slides on slide warmer (Fisher, Cat# 120594) at 37 °C overnight to dehydrate. 

SECTION: PASH Staining
3. Label slides with PASH, date, and additional relevant information.
SECTION: PASH Staining
4. Hydrate slides with de-ionized (DI) water for 5 min 

SECTION: PASH Staining
5. Incubate with 0.5% Periodic Acid from kit for 5 min 

SECTION: PASH Staining
6. Rinse DI water for 5 min

SECTION: PASH Staining
7. Incubate with Schiff Reagent from kit for 15 min 

SECTION: PASH Staining
8. Rinse with DI water for 10 min 

SECTION: PASH Staining
9. Incubate with Harris Hematoxylin from kit for 5 min 

SECTION: PASH Staining
10. Rinse with DI water for 5 min 

SECTION: PASH Staining
11. Quick dip in 0.5% Acid Alcohol from kit (or 20 s)

SECTION: PASH Staining
12. Rinse with DI water for 5 min

SECTION: PASH Staining
13. Quick 2 dips in Bluing solution of 1% Lithium Carbonate (or 40 s)

SECTION: PASH Staining
14. Rinse with DI water for 5 min 

SECTION: PASH Staining
15. Dehydrate using the following series:
75% ethanol 5 min 
75% ethanol 5 min 
85% ethanol 5 min 
85% ethanol 5 min 
95% ethanol 5 min 
95% ethanol 5 min 
100% ethanol 5 min 
100% ethanol 5 min 
Xylenes 5 min 
Xylenes 5 min 
SECTION: PASH Staining
16. Mount with Permount (EMS, cat#17986-01) and air dry in hood for overnight.
SECTION: PASH Staining
17. Image using Virtual Slide System (OLYMPUS, Japan) using 20x, 40, and 60x objectives. 
SECTION: PASH Staining
18. Save images as tiff files with clear labeling: Eye ID/L, R_Age/Gender_Slide #_Dye_magnification (e.g.: 1234567L_97F_050_PASH).