I have done IHC before, but only with tissue samples. I was wondering if the protocol is same for tissues and cultured cells and what precautions I would need to take, while staining monolayer cells.
Hi Manasi this is really easy to do. Why dont you send me an email and I will send you a protocol. It is essentially the same as doing IHC for sectioned tissue.
Quincy Quick, PhD
Dear Manasi,
It is pretty much the same principles. With monolayers you can significantly reduce the fixation, washing and incubation times. For example, 15-20 mins fixation in 4% PFA is sufficient; primary and secondary antibody incubation times can be reduced to around 2 hours; and PBS washes can be reduced to 10 minutes each. Reduce the concentration of detergants, if you have to use them to permeabilize the membranes. You also need to be gentle when changing solutions as it is easy to disturb/uplift/loose the cells. Also be careful, when placing a coverslip, I usually build a protective wall around the cell to prevent the weight of the glass to smash the cellular structures.
Best wishes,
Refik
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