Immunoperoxidase staining of frozen sections with Vectastain kit.

DRH and DJ 9/10/93 Friday at CBRC

 

•Wash 3x PBS,. 5 min each wash.

•Soak in blocking serum from kit(yellow bottle) for 20 min.

•Wash 3x PBS, 5 min each wash.

•Put on 1o antibody for:

37o C for 30 min or

R.T. for 11/2 hour or

O/N at 4oC.

•Wash 3x PBS, 5 min each wash.

•Put on 2o biotinylated antibody (blue bottle) for 30 min at r.t..

•Wash 3x PBS. 5 min each wash.

•Incubate in vectastain reagent (grey bottle) for 30 min.

•Wash 3x PBS. 5 min each wash.

•Incubate in peroxidase AEC (from kit) approximately 10 mins, more or less depending upon staining intensity and desired intensity.

•2 quick washes and then one 5 min wash in tap water

•Counterstain with hematoxylin 1 or 2 dips.

•Wash 3x water. 5 min each wash.