I didn't see an avidin/biotin block but noticed that your donkey anti-goat
has biotin as its label. Do you think that a blocking step might clear up
the background on the ones w/ the strong signal (because of endogenous
biotin in the brain getting stained)?
Or maybe it would be easier to clean up the staining if you tried something
in the range of 1:600- 1:1000 for that biotin labeled seconary.
Just some thoughts,
On Wed, Sep 17, 2008 at 11:12 AM, Caroline Bass wrote:
> 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
> 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.
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