Why my BrdU staining shows so many false positive?
My protocol was:
The hippocampal slices were rinsed with 0.05 M Trisbuffer (TB) incubated for 5 min at room temperature with 0.3% H2O2, denatured by incubation for 30 min at 37◦C with 2 N HCl, and blocked by incubation for 1 h at room temperature in 10% normal goat serum (Sigma, USA) in TB containing 0.5% Triton-100. They were then incubated overnight at 4◦C with mouse monoclonal antibody against BrdU (1:100 in TB; Cell Signal, USA) and incubated sequentially for 1 h at room temperature with biotinylated horse anti-mouse IgG (H + L) antibodies (1:200) in TB; Vector, USA), 30 min at 37◦C with streptavidin-horseradish peroxidase (1:300 in TB), and 10 min at room temperature with3,3-diaminobenzidine tetrachloride (Sigma), then dehydrated in ethanol and xylene.
I think you're developing your DAB for too long, as evidenced by the hazy brown background in the tissue. 5 minutes development is typically the longest I'll use even in FFPE sections.
You may also want to try a stronger peroxide quenching step. You should also include some peroxide during DAB development (though I presume you have done this).
I assume you are working with mouse hippocampal sections, if this is the case then try to use primary antibody raised in any species other than mouse. Anti-mouse secondary will bind with endogenous mouse IgG and gives false positive signals. As John said, DAB development step can be shorten to couple of minutes, 10 minutes are definitely too much.
Hi,
I agree with Rahul Kumar
In addition, I would suggest to:
1. shift H2O2 step to just after the secondary an. and just before the ABC kit step and prolong incubation upto 20 minutes.
2. Dilute your antibodies (primaries and secondaries) in the blocking buffer half diluted.
These two steps will significantly cut the off target signals.
Good luck
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