[Histonet] RE: Confusing IHC results?
When antibodies are applied at a far too high concentration staining
will get weaker. This is called the "prozone effect". I would
suggest to go up with your dilutions like: 1:2000 - 5000 - 10,000 -
20,000 - 50,000. At least until the specific staining disappears
again. Lately, I came across the same situation and ended up
unexpectedly at 1:200,000-500,000!
Stupid remark perhaps, but did you take care that your blocking serum
isn't normal goat since your secondary is anti-goat?
As far as the reward: I keep it to the drink!
Chris van der Loos, PhD
Dept. of Pathology
Academical Medical Center M2-230
NL-1105 AZ Amsterdam
phone: +31 20 5665631
fax: +31 20 6960389
Date: Tue, 19 Jul 2005 17:11:13 -0400
From: "Trajkovic, Dusko"
Subject: [Histonet] Confusing IHC results?
I just completed an IHC run for Calpain 1 (Santa Cruz), using
1:800, 1:1000 and 1:2000. I also ran previous runs where the dilutions
1:100, 1:200, 1:400 and 1:800. I am using a Universal secondary from
at 1:200 on all of the above slides.
My staining results do not vary at all. As a matter of fact, my 1:2000
more intense and has more non specific staining than my slide at
When I run my negative at any of the concentrations to match my
dilution, it is completely clean.
What is going on here? Has anyone ever experienced something like
Primary is a goat polyclonal. I have tried other secondaries such as,
anti goat, horse anti goat, rab! bit anti
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