IHC in Cartilage - A little help to keep me from pulling out my hair? (Jun/29/2010 )

Hi all,

I am attempting (unsuccessfully, as you may have guessed) to perform anti-caspase IHC on paraffin-embedded mouse cartilage. I am studying apoptosis in fracture calluses, and have previously gotten real-looking positive results using TUNEL, but have been unable to successfully perform IHC to confirm my results. I either have a maddening excess of background or no stain at all, and after tweaking pretty much all of the steps, I am at a loss as to what I should do.

Here are the conditions in which the tissues are prepared:
-Fixation in 4% PFA overnight
-Decalcification in 19% EDTA for 4 weeks at 4C then embedded in paraffin blocks
-Sectioned at 10um thickness
-I'm using 2 different polyclonal rabbit anti-caspase antibodies, the goat anti-rabbit secondary kit from vector (catalog number pk-6101), and the DAB kit from vector.

I have tried various dilutions of both the primary (1:25 up to 1:500) and secondary, various DAB incubation times, antigen retrieval with Proteinase K (37C for 15min), antigen retrieval with citrate buffer, blocking, no blocking, jumping 5 times on my left foot and spinning clockwise 3 times, praying to the IHC gods, and talking to my sections pleadingly, but nothing has worked.

Any ideas?

Thanks!
Marie

I have not done work in that area but may I ask if the anti-caspase method works on a different tissue when a positive and negative are run side by side?

sgt4boston on Jun 30 2010, 03:27 AM said:

That's one of the most frustrating things! When I run the same protocol at the same time on skin, I get a high background in the skin when I get no stain at all in the fracture callus.