[Histonet] Doublecortin staining with Abcam antibody

[Montina Van Meter]

Tifei,
   I routinely section rat brain at 40 microns and free-float them through the immuno protocol.  Here are a few 
   suggestions:
 
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 
    TBS rinses.
 
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 
     secondary antibody.
 
 
Hope this helps,
 
Tina
 
 
 
Montina J. Van Meter
Lab Manager
Pennington Biomedical Research Center
6400 Perkins Rd.
Baton Rouge, LA  70808
225-763-2564

________________________________

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.



2009-04-24



TF
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