[Histonet] Doublecortin staining with Abcam antibody
Montina Van Meter
Montina.VanMeter <@t> pbrc.edu
Fri Apr 24 10:33:50 CDT 2009
I routinely section rat brain at 40 microns and free-float them through the immuno protocol. Here are a few
1. Did you perform a dilution series to optimize the best antibody concentration?
2. I antigen retrieve difficult antibodies (especially with thick sections) in a Decloaker (Biocare), thus opening up
the binding sites.
3. I'm assuming the tissue is fixed - we incubate the sections in a 1% sodium borohydride solution (after
Decloaker step) to rid excess aldehyde in tissue prior to the blocking step (20-30 mins.) Follow with PBS or
3. We also use 0.3% triton - this is not a high concentration for 40 um sections.
4. I use 0.3% triton in my antibody diluent - overnight incubation.
5. Make sure you have at least 3 rinses in between incubations to rid nonspecific "glow garbage" from the
Hope this helps,
Montina J. Van Meter
Pennington Biomedical Research Center
6400 Perkins Rd.
Baton Rouge, LA 70808
From: histonet-bounces <@t> lists.utsouthwestern.edu on behalf of TF
Sent: Fri 4/24/2009 5:25 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Doublecortin staining with Abcam antibody
hi all, I am staining 40 um brain frozen sections with Abcam anti-DCX (rabbit source).
With 10% goat serum blocking for 1 hour before primary antibody, I observed a lot of non-specific fluorescence on my slide. Anyone experienced this before?
Second, I did not perform the antigen retrieval before the staining. The sections are made on a microtome, and floating sections are then mounted on slides.
But I added 0.3% triton to the primary antibody dilution. Will very long incubation (1-2 nights room temperature) with high concentration of triton affect the staining result? Such as "leaking" pattern of staining.
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