Wholemount immunocytochemistry for RyR.
All the solutions are kept and used at 4oC. Incubations and washes are performed at 4oC with gentle agitation on a flat bed rotary shaker.
1) Dissect tissue in PBS (Sigma) and transfer tissue to a 1.5 ml eppendorf tube.
2) Fix tissues in 100% Methanol for 5 minutes at -20oC
3) Wash 3 times for 10 min. in PBS.
4) Fix again in PBS containing 2% Paraformaldehyde + 0.1% Glutaraldehyde for 20 min.
5) Wash 3 times for 10 min. in PBS.
6) Wash 5 times or more over a period of 2 hours in PBT (PBS, 0.1% Triton X-100).
7) Block in PAT ( 0.5% BSA, 5% Goat serum, 0.1%Triton in PBS) for 1 to 2 hours.
8) Add affinity-purified RyR antibody diluted 1:100 in PAT and incubate overnight at 4oC.
9) Wash all day in PBT changing solution every hour.
10) Block in PAT for 1 to 2 hours.
11) Add secondary antibody (Jackson Immunologicals: Texas red-conjugated affinity-purified goat anti-rabbit antibody diluted 1:500 in PAT), cover tubes with silver foil and incubate overnight at 4oC.
12) Wash 3 times for 20 min in PBT (Go to step 6 if more than 1 antibody is going to be used)
13) Wash 4 times for 1 hour in PBS (All day if you can gives better results)
14) Optional. Incubate with DAPI and 3 x5 min washes with PBS.
14) Mount slides. We use Vectashield mounting medium.
Good luck!!
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