I need to do double IHC on FFPE mouse tissue. Unluckily two antibodies I have to use are all rat anti-mouse. The protocol for these antibodies are very similar: Pro K digestion, Primary antibody, Rb anti-Rat 2ND antibody and the rest is following PerkinElmer TSA biotin system instruction (Streptavidin-HRP, biotinylated-tyramide and Streptavidin-HRP).
I will perform the two IHC sequentially. I have two questions:
1. Do I need to do Proteinase K for the second antigen?
2. Can I keep all other steps the same and just use Streptavidin-FITC or Streptavidin-Texas Red at the final step to differentiate two antigen?
BTW, could you recommend a good website to learn double IHC?
Any input is highly appreciated!
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