Label slides with antibody and treatment to be used.

De-paraffinize slides in fresh xylenes, then in a descending ethanol series.

De-paraffinize slides 0h 5m 0sin fresh xylenes. (1/2)

De-paraffinize slides 0h 5m 0sin fresh xylenes. (2/2)

De-paraffinize slides in ethanol 100% for 0h 1m 0s.

De-paraffinize slides in ethanol 100% for 0h 1m 0s.

De-paraffinize slides in ethanol 95% for 0h 1m 0s.

De-paraffinize slides in ethanol 80% for 0h 1m 0s.

De-paraffinize slides in ethanol 70% for 0h 1m 0s.

Formic Acid Retrieval (If necessary, do here).

Immerse slides in ddH₂O for 0h 1m 0s, place in recycled FA for 0h 5m 0s and wash in running tap H₂O for 0h 10m 0s.

Microwave antigen retrieval (CA; If necessary do here).

Dilute antigen unmasking solution (Vector Labs, citric acid) 1:100 in dH₂O (2.5mL/250mLdH2O/boat).

Place in Biogenex EZ-Retriever microwave for 0h 15m 0s at 95°C.

Cool for 0h 20m 0s at95Room temperature.

Wash slides for 0h 10m 0s in running tap H₂O.

Immerse in freshly prepared Methanol/H₂O₂ (150mLMethanol + 30mL stock 30% H₂O₂) 0h 30m 0s.

Then, wash in running tap H₂O for 0h 10m 0s.

Wash in 0.1Molarity (M)Tris buffer, 7.6 0h 5m 0s. Discard all Tris washes.

Block in 0.1Molarity (M)Tris/2% FBS (Tris/FBS) 0h 5m 0s+. Keep blocking solution for up to 2 weeks @ 4°C.

Dilute primary antibodies in Tris/FBS, and prepare humidified chamber(s) by soaking towel in the middle of the slide chamber(s).

Wipe excess fluid off back of slides and from around tissue and apply 200µL of primary antibody to slides. Make sure antibodies cover all sections.

Incubate at 4°C in humidified chamber 0h 5m 0s.

Rinse off antibody from tissue using Tris.

Wash in Tris 0h 5m 0s.

Block in Tris/FBS 0h 5m 0s.

Dilute Vector biotinylated IgG 1:1000 in Tris/FBS and apply200µL to wiped slides.

• For immunofluorescence , dilute fluorescent secondary antibodies 1:500 in Tris/FBS and apply 200µL to wiped slides. Keep slides in the dark from here on. Incubate at 4°C in humidified chamber overnight or at 4Room temperaturefor 3h 0m 0s or overnight at 4°C . Proceed to Day 3.

Incubate at 4Room temperature in humidified chamber 1h 0m 0s.

Rinse biotinylated IgG using Tris.

Wash in Tris 0h 5m 0s.

Block in Tris/FBS 0h 5m 0s.

Mix AB solution (Vector peroxidase standard) in Tris/FBS to a dilution of 1:000 (ie add 1µLof A and 1µL of B to 1mL of 0.1Molarity (M) Tris/2%FBS). Vortex and let sit 0h 15m 0s before use. Then, apply 200µL of AB solution to wiped slides.

Incubate at 4Room temperature in humidified chamber for 1h 0m 0s.

Rinse off AB using Tris.

Immerse in Tris 0h 5m 0s.

Make Vector DAB solution (1 drop of DAB per mL of Stable DAB Buffer) .

Apply 200µL of DAB to each slide and incubate until a visible brown signal is seen and well developed.

Rinse with Tris and place in dH₂O. Wash 0h 5m 0s in dH₂O. Filter Harris hematoxylin.

Counterstain briefly with Harris hematoxylin (~0h 0m 15s, depending on age).

Wash in running tap H₂O 0h 5m 0s.

Dehydrate and clear in ascending ethanol and xylenes.

Dehydrate and clear in 70% ethanol for 0h 1m 0s and 70% xylenes for 0h 5m 0s.

Dehydrate and clear in 80% ethanol for 0h 1m 0s and 80% xylenes for 0h 5m 0s.

Dehydrate and clear in 95% ethanol for 0h 1m 0s and 95% xylenes for 0h 5m 0s.

Dehydrate and clear in 100% ethanol for 0h 1m 0s and 100% xylenes for 0h 5m 0s.

Dehydrate and clear in 100% ethanol for 0h 1m 0s and 100% xylenes for 0h 5m 0s.

Coverslip with cytoseal.

Dry in tissue processor closet 0h 5m 0s.

Filter Sudan Black. This process can take a long time, so start early.

Rinse off AB using Tris.

Wash in running tap H₂O for 0h 5m 0s.

Wash in Tris for 0h 5m 0s in green boats.

Sudan Black Treatment (0.3% Sudan Black B in 70% Ethanol)

Use a control slide (usually 1 positive primary and 1 secondary only) to titrate for background reduction without changing signal intensity (usually 0h 0m 10s to 0h 1m 0s).

Image before and after Sudan black treatment for various times.

Treat all slides identically.

Wash in 0.1Molarity (M) Tris 0h 5m 0s in green boats.

Coverslip using non-photobleaching reagent (FluorMount with DAPI). Allow to dry completely before imaging on scanner.