ABC Kit For Visible Chromogen

Protocol Overview

1) Incubate tissue in PBS solution containing 0.3% H2O2 for 5 min at room temperature to inhibit endogenous peroxidases (H2O2 stock is 30%). 2) Remove H2O2 solution and rinse tissue once with PBS solution.3) Incubate tissue in 5% BSA (in PBS) solution to “block” nonspecific antibody binding for 1 hr at room temperature. 4) Incubate tissue in desired primary antibody for overnight.5) Remove primary antibody and rinse tissue 4 times, 10 min each with PBS-BSA solution.6) Add biotinylated secondary antibody to tissue for 2 hr at room temperature.7) Prepare PBS containing avidin/biotin solutions from VECTASTAIN ABC kit. 8) Remove biotinylated secondary antibody and rinse 4 times 10 min each with PBS solution. 9) Add PBS/avidin/biotin solution for 2 hr at room temperature. 10) Remove PBS/avidin/biotin solution and rinse 4 times, 10 min each with PBS.11) Prepare VECTOR chromagen according to manufactures protocol.12) Mount and dehydrate tissue and coverslip using Permount.