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IHC: antibody titration - determine the right dilution (Nov/03/2010 )


I have to stain my tumor xenograft sections with Ki-67 from BD (cat no. 550609). The antibody datasheet suggests to try 1-10 to 1-50 dilution. I have tried 1:20, 1:40, 1:50, 1:100 and 1:400 and all them seem to give similar staining intensity. Shall I try 1:800 and 1:1000 dilution?



why not? A good starting range for a first go is serial 1:10 dilutions then you can narrow your focus.


Are you sure that the staining you are seeing at the moment is specific? What controls are you doing? BD is normally pretty good for protocols and dilution advice for their antibodies, so if you are using it at a much greater dilution than that suggested, you may be doing something wrong.



I would be highly indebted to anyone who can tell me where I'm going wrong. I have been strugling wth IHC for the past 2 months. I have tried bpcatenin antibidy from BD suing 1:100, 1;200, 1:500 dilution and they all stain with the same intensity? I have tried a control w/o the primary antibody but with the secondry antibody and I do not get any false positive staining.

I'm simply dilution the primary antibody in 3% BSA. If I do a 1:100 dilution, I add 1ul antibody to 99ul blocking solution?


Do serial 1:10 ie 10,100, 1000, 10,000 etc at some point you should see a drop off then focus on that area. If 2ndary antibody is not causing false positives and your primary is specific you should be able to get the right titer.