I tried performing WB to detect PKC-e in PBMC lysate but did not get good results. I wish to perform IHC for the same protein using PBMCs. It would be of great help if someone could suggest a protocol for performing IHC using PBMCs.
Thank you in advance.
Yes, smear the silde as you would a normal blood film, air dry and incubate with antibodies of interest. Then process like with MCDH1 dyes (May - Grunewald type stain) for contrast
Kevin Marron Thank you very much for your answer.
As I am entirely new to the technique/IHC, is it possible for you to share the protocol?
Thank you in advance
Hi Nikhita. So we are thinking outside the box here a bit. Are any aspect of the PKC proteins expressed on the cell surface? But in theory if you air dry the whole blood film, or a similar smear of PBMC's from say a buffy coat. You would then add your diluted IHC antibody to bind to the epitopes, incubate and wash off. (you might need to permeabilise the cells if proteins are intracellular and maybe fix with alcohol so they don't float off with the wash step) Then visualise with the second part of the IHC kit, usually streptavidin-biotin complex (appears brown under microscope). The kit manufacturer will have instructions for use. Then for some cell contrast do a H&E stain (Haemotoxlin & Eosin). The alcohol step here will definitely fix the cells in place. Coverslip and stick under a light microscope.
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