Hi all, I'm a summer student and the project I was given was to optimize the immunohistochemistry in my lab. BUT I'M NOT GETTING ANYTHING TO WORK!! I've been told it always worked before....So I was wondering if anyone has any ideas or protocols that work? I'd really appreciate any help I can get! Here's the protocol I'm using: our slices are 250-300 um thick. 1) I leave them in formalin for 2 days for the fixative step. 2) Wash slices 3X (15 min each) in PBS-T (PBS with 0.3% tritonX100 to help permeabilize my slices since they're thick) 3) Blocking step for 1/2 hr with 2%normal horse serum in PBS-T 4) Incubate in 1o antibody at 1:1000 in PBS-T + 2%normal horse serum for 72 hrs at 4C with shaking 5) Wash slices 5X in PBS-T 6) Blocking step for 1/2 hr with 2%normal horse serum in PBS-T 7) Incubate at room temp with 2o antibody at 1:500 +2%normal horse serum in PBS-T for 2hrs with shaking and kept in the dark 8 ) Wash 5X (1/2 hr each) in PBS before mounting Terri _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet