AW: [Histonet] Confusing IHC results?

From:"Gudrun Lang"

Do you use the PAP-technique? I have read, that in some circumstances in the
lower dilution the antibodies are so many, that the bridge-antibody is bound
with both fab's and there is nothing left for the PAP-complex (bridge doesnt
work). So staining will be weaker than in a higher dilution. Just a thought.


-----Ursprüngliche Nachricht-----
[] Im Auftrag von Trajkovic,
Gesendet: Dienstag, 19. Juli 2005 23:11
An: ''
Betreff: [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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