There are a lot of things you could try, but I'd
recommend, as a first shot, that you omit the
Trition from the rinses after the primary and
from the secondary solution. I don't understand
exactly why, and it's certainly not
intuitive, but for quite a few primaries,
including Triton at these steps can dramatically
increase background staining. I'd be surprised
if changing the secondary concentration improved
things, but it's quite possible that reducing the
primary concentration might have a beneficial effect.
>Date: Wed, 17 Sep 2008 14:12:19 -0400
>From: Caroline Bass
>Content-Type: text/plain; charset="ISO-8859-1"
>Sorry if this is a double post but I didn't see my first one in the digest.
>Iım hoping someone could help me troubleshoot my staining protocol. I am
>staining to EGFP in microglia (produced by a virus I injected in the brain).
>Hereıs my general protocol:
>Deactive endogenous peroxidases (0.1M PB + 20% methanol + titon-x100 (80 ul
>in 40 ml total) + 0.3% h2O2) for 15 min.
>Wash 3X15min in 0.1MPB+0.3%titon
>Incubate in 0.1MPB+0.3%triton+1%normal donkey serum for 1 hr
>Incubate with primary (goat anti-GFP from Rockland) overnight at 4 degrees
>Wash 3x15 min 0.1M PB + 0.3% triton
>Incubate in with secondary (Jackson Donkey anti-goat, biotin labeled)
>overnight at 4 degrees (1:500)
>Wash 3x15 min in 0.1M PB
>Use ABC kit and vector SG for substrate (canıt remember how long I let it
>develop, but it was within a couple of minutes).
>So, the bottom line is that I did a test section and I thought it had a lot
>of background, so I incubated the other sections for less time. However,
>when I took a closer look, the sections with little background had no
>microglial signal, while the one with high background had a very clear and
>noticeable signal in the place it should be. So, the question is how to cut
>back on the noise. Would increasing the secondary concentration to 1:1000
>help? Iım following a protocol someone else came up with, but it seems
>strange that the secondary is at a higher concentration than the primary.
Dr. David Wirtshafter
Laboratory of Integrative Neuroscience
Department of Psychology, m/c 285
University of Illinois at Chicago
Room 1009 Behavioral Sciences Building
1007 W. Harrison St.
Chicago, IL 60607-7137
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