I plated out cells onto cover slips in a 24 well plate overnight
Next day I washed 3 times for 5 minutes each with tbs-tween
Then used blocking solution of 1x BSA in TBS for an hour
After that I washed again, then applied 200 ul of my primary antibody which was diluted with the same blocking soln (Overnight in cold room)
Next day, wash again with tbs-tween 3x for 5 min
I applied the secondary antibody for 1.5 hours at room temp
Applied the DAB solution and let it sit for 15 minutes (saw no changes in color).
It was the same procedure for the Fluorescence except, my secondary antibodies were not HRP, but fluoro