[Histonet] Troubleshooting IHC

Adam . anonwums1 <@t> gmail.com
Thu Mar 25 10:43:28 CDT 2010

Hi all,

I've recently run into a problem troubleshooting IHC on mouse bones. I am
using the tyramide amplification system using a goat primary, anti-goat
biotin, strepavidin-HRP, biotinyl tyramide, followed by another
strepavidin-HRP and then DAB+ from Dako. When I use an isotype goat IgG, I
get essentially no staining.

I titered it once for immunofluorescence (SA-fluor instead of HRP in the
last step) and got a titer of 1:800, and these staining conditions are quite
reproducible. Then I titered for IHC, and 1:200 gave the best signal to
noise. I did a batch of staining using those IHC conditions, and it stained
beautifully, comparable to my IF. I then tried to repeat it on a second
batch of sections processed in the same way, and the background was terrible
(but not on my isotype slide). I thought maybe it was a problem in
processing so I did a third batch of sections, and the background was still
really bad. I see real staining sometimes, but I need to quantify this
staining using histomorphometry, so I really need clean staining. Any ideas?
The only thing I can think of is that 1:200 is just at the limit of
titration that gives too much background.


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