I am doing IF on deparaffinized mouse tissues, (original tissues, are vertical cut mouse tissue sections ; mouse was cut vertically several times, side by side, and tissues were paraffinized). Could someone please recommend some proteins that I can detect in these tissues, and that are constitutively expressed (The HSP90/Alpha-tubulin IF counterpart of Western Blot). Thing is I wanna make sure that the protocol is working fine before proceeding with my protein detection, since my first try wasn't successful. Is phalloidin a good choice in that case? Many thanks for your time.
Just a side question, is gelatin in PBS a suitable blocking buffer for IF. In what ratios please, thanks.
Is this immunohistochemistry? If the mice are adult, I would probably choose a connective tissue marker such as alpha smooth muscle actin (it labels blood vessels). I would recommend asking colleagues who work on mice to let you try any antibody which may work well on mouse tissue. If you have all the organs represented in your section, you could choose a protein which you know will be expressed in a specific organ. For embryos - I did IHC for GATA1 once which is strongly expressed in liver. For in situ hybridisation I would recommend beta-actin as a general marker and this might work for IHC.
Hi Moe.
I totally agree with Jennifer.
Plus: In paraffin sections you maybe have to perform antigen retrieval protocols until your antibody is able to bind to the antigen. Some companies publish detailed staining protocols in their datasheets and on their websites.
Answer to the "side question": If you perform immunohistochemistry, then you should use the serum of the animal in which the secondary antibody was made as blocking buffer in PBS with or without Triton. If the antigen is intracellular then you should use Triton to make the membrane permeable for the antibody.
We use 10% Serum in PBS with 0,1%TritonX-100.
Good luck!
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