What types of mouse tissue are you working with? Are they fresh frozen with post-fixation or fixed frozen? Do you block biotin? Do you have an isotype control and a no primary control? I am thinking biotin's the likely culprit as the kit doesn't come with the very necessary biotin blocking reagents ... Andrea Hooper
--- On Wed, 2/24/10, Nicholas David Evans <ndev...@stanford.edu> wrote: From: Nicholas David Evans <ndev...@stanford.edu> Subject: [Histonet] M.O.M frozen section background To: "histonet" <histonet@lists.utsouthwestern.edu> Date: Wednesday, February 24, 2010, 6:33 PM Hi All, Does anyone have any tips on reducing background staining when attempting to detect mouse monoclonal antibodies on mouse tissue? We currently use Vector's M.O.M kit - it works great on PPFE tissue with nice specific staining after pepsin unmasking, but in frozen tissue the background is much higher than the signal. I routinely use 4% PFA pre-fix, then 3% H2O2 in MeOH for 3 mins, then all the blocks that come with teh kit. I am using several different cytokeratin Abs on 10 um frozen mouse sections. The only thing that I could think to try was to dehydrate the tissues, rehydrate and try again with the unmasking, but if anyone has a better idea or commonly deals with this problem, please let me know. Best wishes Nick _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet