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I ordered Anti-PDX1 antibody (ab47308) two weeks ago. I was testing the antibody for immuno-fluorescence staining using 1:100, 1:1000 and 1:10,000 dilution however none of them worked. Cells were fixed with 10% PFA for 10 mins at RT. it is unlikely the secondary antibody since it worked well for other primary antibodies. Also, when I received the antibody, there was precipitates in the solution. Could you please help me with this issue? Any suggestion will be appreciated.
Asked on Oct 07 2011
Following up my previous message, the laboratory and I think the problem may be the 10% PFA fixation for 10 minutes at room temperature. The lab used 4% PFA and fixed for 2 hours at 4C. I think the fixation time could be made shorter than two hours, for instance 30 minutes at 4C. This will preserve the structure of the cell, but will hopefully minimize the cross-linkages introduced by the PFA that may be hindering antibody:antigen recognition. The acetone/methanol approach is an alternative, which does not create chemical modifications of the PDX1, but the laboratory has not tried this. Your current fixation protocol may be fine for other antibody stains, but each antibody:antigen interaction is unique, and ab47308 may have difficulty with 10% PFA at RT. Please let me know how the antibody works for you if you try one of these fixation modifications, and please contact me if you have any questions.
Answered on Oct 07 2011