[Histonet] M.O.M frozen section background

[Andrea T. Hooper]

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 <ndevans <@t> stanford.edu> wrote:


From: Nicholas David Evans <ndevans <@t> stanford.edu>
Subject: [Histonet] M.O.M frozen section background
To: "histonet" <histonet <@t> 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




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