[Histonet] thick slice IHC help!!

[Terri Waller]

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