Antigen Unmasking
Bring slides to a boil in 10 mM sodium citrate buffer pH 6.0 then maintain at a sub-boiling temperature for 10 minutes. Cool slides on bench top for 30 minutes.
Wash sections in dH2O three times for 5 minutes each.
Wash section in wash buffer for 5 minutes.
Block each section with 100-400 ul blocking solution (1X PBST, 5% goat serum) for 1 hour at room temperature.
Remove blocking solution and add 100-400 ul primary antibody diluted in 1X PBST, 5% goat serum to each section. Incubate overnight at 4C.
Remove antibody solution and wash sections in wash buffer three times for 5 minutes each.
Add 100-400 ul biotinylated secondary antibody, diluted in 1X PBST, 5% goat serum. Incubate 30 minutes at room temperature.
Remove secondary antibody solution and wash sections three times with wash buffer for 5 minutes each.
Add 100-400 ul Striptavidin-HRP reagent to each section and incubate for 30 minutes at room temperature.
Wash sections three times in wash buffer for 5 minutes each.
Add 100-400 ul DAB substrate to each section and monitor staining closely.
As soon as the sections develop, immerse slides in dH2O.
Wash sections in dH2O two times for 5 minutes each.
Mount coverslips.
