I am trying a multiplexing IHC, and we don't have any microwave stronger than 800W, and I am not sure if this could be an issue. Also, is it possible to do any of the steps Overnight?
Thanks
it would be better if you do primary antibody incubation for overnight. for microwave, you just have to boil discountinuously for a few minutes.
I agree with Arun. Primary ab can be o/n and 1000W is not needed. We use for example 4x5 minutes 440W (it should boil, but not too hard). For each ab we optimize the time needed (2-4 x5 min).
Most IHC steps (except permeablization steps) can be left overnight (or longer) at 4C (depending on how well your tissue is fixed). Any buffer rinse step can be left as well. It is best to leave the primary antibody in overnight in your blocking solution at 4C to give it the best chance of binding without introducing too much non-specific binding (more common when left at RT).
Hallo Almudena, not every primary antibody needs antigene retrievel. I recommand to run a delution raw to find out the optimal concentraton of your ab. I prefere incubaton of primary ab over night at room temperature becaus higher temperature forcecs the antibody binding and that leads to lower ab concentration. Several blocking steps are necessary to prevent unspecific background staing. For example blocking of endogeneous peroxidase when you are working with peroxidase labelled secondary abs or other detectiosystems like ABC or Streptavidin. If you are working with ABC or Streptavidin also a avidin/biotin block is important. detergences like Triton or Tween 20 are helpfull for better ab penetration.
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