Hi, I have been trying to immunostain Hela cells on coverslip with HSP 70 Ab with out any success..The protocol I am using include fixation with methanol (-20 degree) for 10 min, permeabilization with .5% Triton X-100 for 15 min, blocking with 1% BSA for 30' and incubation with Ab followed by Sec Ab for 1 h each. Each step is followed by a 15 min PBS wash. I'm unable to figure out where I'm going wrong as western blot works out fine under similar cond of heat shock. Any suggestions..
hi
this may be helpful
www.activemotif.com/documents/1564.pdf
http://openwetware.org/wiki/Silver:_High-throughput_immunostaining
regards
reduce the time in methanol (as its overfixed if u keep it for more time)and also reduce the conc of triton X , may be you can try 0.1 %