Immunostaining of Heat shock proteins

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rbb2007
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Immunostaining of Heat shock proteins

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..

Kvachhani
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hi
Arvind Singh Pundir
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reduce the time in methanol

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 %