[Histonet] Confusing IHC results?

Joe Nocito JNocito <@t> Pathreflab.com
Wed Jul 20 08:09:23 CDT 2005

some suggestions would be to titer out your secondary antibody further, say
1:400 and 1:800, and/or titer Calpain 1 out to 1:4000 and 1:8000 you might
have to perform a checkerboard titration. i.e.
Calpain 1 1:4000 Secondary 1:400 & 1:800

Calpain 1 1:8000 Secondary 1:400 & 1:800

I know it's a pain to titer out your secondary when it works on other
antibodies, but I had to do this when I was at the AFIP some time ago. Just
some thoughts.

Joe Nocito, BS, HT(ASCP) QIHC
Histology Manager
Pathology Reference Lab
San Antonio, TX

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu]On Behalf Of
Trajkovic, Dusko
Sent: Tuesday, July 19, 2005 4:11 PM
To: 'histonet <@t> lists.utsouthwestern.edu'
Subject: [Histonet] Confusing IHC results?

Hi everyone,
I just completed an IHC run for Calpain 1 (Santa Cruz), using dilutions
1:800, 1:1000 and 1:2000. I also ran previous runs where the dilutions were
1:100, 1:200, 1:400 and 1:800. I am using a Universal secondary from vector
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 looks
more intense and has more non specific staining than my slide at 1:100.
When I run my negative at any of the concentrations to match my primary
dilution, it is completely clean.
What is going on here? Has anyone ever experienced something like this.
Primary is a goat polyclonal. I have tried other secondaries such as, bovine
anti goat, horse anti goat, rabbit anti goat, donkey anti goat, with either
negative results or sections staining completely brown.
Any suggestions, advice, email lashings would be greatly appreciated. Person
to come up with a solution will receive a hug and a kiss, OR a couple of
drinks at the bar, during the NSH symposium. It's your choice.
Hell, you can take a chance and go for both.
See, I'm going crazy trying to figure this out!!!
Thank you,
Dusko Trajkovic

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