Ki67 Antibodies - (Aug/20/2007 )

Hey all-

I'm looking into purchasing a Ki67 antibody to use in mouse tissue and have a few questions someone may be able to help me out with. Ideally I'd like an antibody that works in paraformaldehyde fixed tissue that is frozen and cut in OCT. Has anyone done and IHC with a Ki67 antibody in frozen tissue? Most of what I read about Ki67 IHC is done in paraffin/formalin fixed tissue. Another question that I have is whether the Heat Induced Epitope Retrieval is required because one is using a Ki67 antibody, or because the antibody is used on paraffin embedded sections. Essentially I'm wondering if I need to perform any kind of epitope retrieval on paraformaldehyde fixed/frozen sections.

Thanks in advance for your help.

GronkFix

-GronkFix-

QUOTE (GronkFix @ Aug 20 2007, 08:03 PM)

Hi,

I used to do some Ki67 stainings in PFA-fixed cryosections (14micron). the antibody I used was rabbit-anti-mouse Ki67 (1:50-1:100 dilution) from DAKO, I usually pre-treated the sections by 2x4min boiling (microwave!) in 0.01M citrate buffer/0.5% Triton X. Works nicely.

Have fun!
dedee

-dedee-

because its frozen you can get away with fixing in acetone, methanol, acetone /methanol(50:50) or the para and the er need only be 0.2% triton x for 10 min (boiling frozen sections tends to make them fall off as you cant stick them in the oven - apes (tespa) is handy as a glue too)

dom

-Dominic-

actually, you don't need any triton x in the boiling buffer at all, it works with only citrate as well. I just "accidentially" prepared a stock with triton once, and used it. but gronkfix is right, if you have kind of sensible tissue, I'd try without boiling.

-dedee-

i meant use the triton x cold on the slides for ten minutes - no hier required - proteinase k is another alternative

dom

-Dominic-

Thanks for the help guys. I suspected that the HIER was required because of the sample prep. I think that I'm going to give the TEC-3 Rat-anti-Mouse Ki67 clone from DAKO a shot with as little epitope retrieval as possible. I'm hesitant to do any kind of boiling because I'd really like to preserve as much of the tissue structure of possible (mouse brain)...I'll start with just the triton. Has anyone had any problems with the DAKO TEC-3 clone?

Thanks again.

GronkFix

-GronkFix-

if your after the least er possible saponin might be worth a try

we use mm1 & ncl-ki67p from vector (ones rabbit, one mouse) and they work fine

dom

-Dominic-