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Intracellular staining of cytokines... crossreactivity? - (Dec/18/2013 )

Hi everyone, 
In June 2012 I bought a number of antibodies from AbD Sertotec and, according to the data sheet from the STAR9B conjugate dated June 2012, the recommended working dilution should be between 1:200 and 1:500. but, in a later datasheet (from november 2013) the recommended working dilution is 1:25-1:100. 
My student asked the Serotec technical support and they told her to use the later STAR9B dilutions (2013) for our flow cytometry experiments: Bovine PBMC surface stained with purified Mo anti-Bov CD4+ Rabbit anti-mo IgG F(ab´)2-FITC and an intracellular staining with mo anti-bo IFNg-PE.
We have got signals from IFNg in every sample and controls. We ran a test with a mo anti-bo CD4 FITC + mo anti-bo IFNgPE; and another test blocking with normal mo serum after de STAR9B staining and the stain with mo anti-bo IFNgPE, and the effect dissapeared, suggesting that the STAR9B is capturing the second conjugate as well... Is that possible???  Is it a matter of wrong dilution of STA9B conjugate and monovalent binding of STAR9B to de primary antibody?
Please, help me to solve this mystery....
Thanks in advance


The problem is that you are trying to use multiple mouse antibodies for detecting different things - the first one you stain with will pick up any subsequent secondary antibodies used.   Ideally you should use different species primary antibodies to detect the different antigens (e.g. mouse anti bov CD4 and rabbit anti bov STAR9b).  If you can't do this, you need to play very very carefully with the conditions to ensure that all the first lot of primary are completely bound by something (labeled secondary or non-specific IgG), so as to prevent them binding any subsequent secondaries.


Another solution is to use directly conjugated primary antibodies, which eliminates this problem but also costs more.