IHC with survivin - (Jun/25/2007 )

Hi all,

I'm doing IHC with paraffin blocks of human esophageal tissue using survivin antibody from cell signaling tech. I can stain the tumor cells nicely but for the epithelium, I cannot stain the cells either in basal layer of non-tumor epithelium or the whole layer of tumor epithelium, as stated in published literature. I have already use 0.1% tween 20 to help permeabilize the section for AB.

Have anyone ever encountered this situation?

Thanks in advance.

Marco

boil the bugger

you might have more luck with heat based epitope retrieval (microwave + EDTA, pressure cooker + citrate buffer etc)
its cos the formalin is a little too good at its job and creates crosslinks every where which need to be broken (and why are you using tween? even triton x would have problems with paraffin blocks)

dom

Thx for your reply.

I have already used citrate buffer (10mM) pH6.0 as antigen retrival agent and boil for 20 mins. Tween 20 is use to allow easy contact between Ab and antigen. I could not stain the cells if I did not add Tween 20 before.

hi

i use 1 mM EDTA (0.05% tween 20) for two lots of five minutes in a microwave - works a treat for caspase related stuff

dom

(oh, and its ph 8)

Did you stain survivin or other caspase related stuff?

Thx in advance.

hi

other - never tried survivin

dom