1. Fill pressure cooker with 1L dI water.  Set pressure cooker time to 16 minutes, high pressure, and cover with lid until water is boiling.
2. Fill two slide holders with Trilogy solution (Cell Marque #920P-10).  Add FFPE slides to one holder.
3. When water boils, add holders to the pressure cooker, close pressure release valve, and seal lid.  When the cooker reaches the correct temp and pressure, the pressurization indicator will pop up and the time will start counting down.
4. When the time has elapsed, open the pressure release valve, and carefully remove the slide holders.  Switch the slides from one container to the other and let sit 15 minutes (hot wash).  Discard Trilogy from first container.
5. Move slides to a container of dI water and rinse.  Trilogy from second container can be saved and reused for the first step of unmasking.
6. Transfer slides to a container of PBS and wash.

1. Deparraffinize and rehydrate FFPE slides by incubating them as follows:

a.     20 min in xylenes (in fume hood)

b.     5 min in 100% ethanol

c.      5 min in 95% ethanol

d.     5 min in 80% ethanol

e.     5 min in 70% ethanol

f.      5 min in 50% ethanol

g.     2x5 min in PBS

1. Heat water in a microwaveable container 10 min at max power, until virtually boiling.
2. Prepare unmasking solution: 1M Tris-HCl, 5% Urea pH 9.5 (60.57g Tris + 25 g Urea in 500 mL water)
3. Place slides in slide holder filled with unmasking solution.
4. Place slide holder in container of boiling water to help control temperature stabilization.
5. Microwave slides at 30% power for 15 min
6. Let cool for 15 min on benchtop.
7. Wash slides in PBS.

1. Blot slides on paper towel and circle tissue sections with hydrophobic pap pen.
2. Refix samples if working with tissue that does not adhere well to the slide: 20 min neutral buffered formalin (10% of 37% formaldehyde stock in PBS), wash 2x PBS. **This step not needed for skin or mammary gland**
3. Enzymatic antigen retrieval with Proteinase K digestion: add Pro K solution 10ug/mL in PBS (1:2000 dilution of stock) to tissue sections and leave on slides for 10 min at RT.
4. Wash 3x in PBST (1x PBS + 0.1% Tween-20).
5. Peroxidase quench, if using tissues with high levels of endogenous peroxidases.  **not necessary for skin or mammary gland**
6. Block slides for 30 min at RT in humidified chamber.  Block= 5% heat inactivated normal goat serum, 1%BSA, 0.01% Triton X-100 in PBS (12.5 mL hiNGS, 2.5 g BSA, 25 ul Triton X-100).  Reserve 5-10mL block for preparing antibody dilutions.
7. Wash 3x with PBST.
8. Incubate slides with primary antibody prepared in blocking buffer(1:200 of previously tested Perp C-terminal antibody fractions OR 1:50-1:100 ECL antibody from Abcam #ab5986) in a humidified chamber for 45-60 min at 37C.
9. Wash 3x with PBST.
10. Prepare secondary antibody dilutions in blocking buffer (1:200 Goat anti-Rabbit-HRP, Jackson Immunoresearch # 111-035-003).  Incubate slides in secondary antibody 45 min in humidified chamber at 37C.
11. Wash 3x with PBST.

1. Incubate slides in diluted DAB buffer (1 drop in 2.5 mL water) for 15 min RT.
2. Aspirate buffer and add DAB reaction mixture (1 drop buffer, 2 drops DAB, 1 drop H₂O₂ in 2.5mL water) for 5-10 min in the dark.  Observe development and stop before stain gets too dark.
3. Remove DAB and save in toxic waste container (including excess prepared DAB solution).
4. Wash slides in water.
5. Counterstain briefly with hematoxylin.
6. Wash in water until hematoxylin stain is gone.  Recover hematoxylin washes to hazardous waste.

 Dehydrate slides by passing through successive washes as follows:

a.     3 min in 50% ethanol

b.     3 min in 70% ethanol

c.      3 min 95% ethanol

d.     3 min 100% ethanol

e.     3 min xylene

19. Wipe coverslip dry with kimwipe.

20. Put a drop of Permount (Fisher #SP15-100) onto slides.

21. Place coverslip on slide, can gently press down to help permount spread and remove bubbles.

22. Let coverslip dry overnight before examining slides under brightfield microscopy.