DAB Staining

Wash freshly sectioned slices of tissue 2-3 times with 1X PBS to remove OCT.

Quench sections for 0h 15m 0s in 3mL of quenching solution (0.1mL 30% H₂O₂, 0.1mL methanol, and 0.8mL 1X PBS).

Wash 4-5 times in 1X PBS.

Block sections in 2mL of 5% goat serum in 0.25% T-PBS for 1h 0m 0s Room temperature.

Stain with primary antibody (pSer129 primary antibody 1:500) overnight at 4°C. Dilute primary antibody in 2.5% serum in 0.25% T-PBS (1mL per well).

Wash sections 4-5 times with 1X PBS.

Transferred sections into secondary solution (1mL of 1% serum in 0.25% T-PBS) for 2h 0m 0s at Room temperature.

Wash sections 4-5 times in 1X PBS.

Transfer into ABC Kit solution (PK4000, Vector Laboratories) containing 10µl of solution A and 10µl of solution B for 1ml of 1X PBS for 1h 0m 0s at Room temperature.

Wash sections 4-5 times in 1X PBS.

Transfer sections to the DAB working solution (SK-4100, Vector Laboratories) under a fume hood. Monitor until it stains well. Well-plate containing sections were gently shaken during staining and the reaction was stopped with transfer to 1X PBS based on dark-signal intensity on the fastest arising staining.

Wash sections 3-5 times in 1X PBS.

Mount sections on microscope slides.

Dry sections 1h 0m 0s at Room temperature.

Dehydrate sections starting with 2 baths of distilled water (~0h 2m 0s).

Wash with 70% ethanol (2 times ~0h 2m 0s).

Wash with 95% ethanol (2 times ~0h 2m 0s).

Wash with 100% ethanol (2 times ~0h 2m 0s).

Wash with 100% xylene (2 times ~0h 5m 0s) to allow section dehydration.

Dry sections at Room temperature (~0h 5m 0s) and covered with DPX mounting medium.