imc-forum.org

Hi all,

As you all know, retrieval is key to staining on FFPE tissues, especially when trying to get 35 antibodies to work well together.

What retrieval conditions have you been having most success with for the broadest range of antibodies and tissues? (Citr/EDTA, temp, time)

I have tried multiple ones including 95C/10 min, 20 min, 30 min; 121C/5 min in pressure cooker. High temp seems to work well for most but some antibodies are still problematic.

It would be very helpful to hear everyone's experience so far.

Thanks,
Santhosh

Hi Santhosh,
I haven't tried anything beyond the recommended EDTA buffer for 10 minutes at 95C. I'm using the Biogenex microwave. I have stuck with these conditions mostly because those conditions are similar to what most antibody manufacturers recommend for IHC, at least for the antibodies I'm using. Of course some do recommend citrate over EDTA likely due to reduced background. I do plan on playing with these HIER conditions again once I can finalize my mouse panels. For IHC, I did previously test abcam's universal HIER antigen retrieval buffer (ab208572) and found it inferior to both EDTA & citrate buffers for a couple of antibodies I tested, but I haven't tried using it for IMC.
-Doug