RE: beta galactosidase immunos

From:"Martin, Ronald"

You emailed me a few of your procedures a few months ago and I am trying to use them as guides. The tissue is perfused (spleen) with a formalin (made from paraformaldehyde), glutaraldehyde and sucrose combo. (so I was told,I don't know much more about that).
I am using the same antibody as your procedure (ICN Biochemical division #55976-monoclonal?) which is purified rabbit anti Galcatosidase along with Vectastain biotinylated goat anti rabbit IgG secondary and Vectastain ABC reagent then DAB.
I have tried straight 3% H2O2, as well as 3% H2O2 made from 30% H2O2 and methanol at different staining points:prior to the primary antibody,prior to the secondary antibody and prior to the the ABC reagent and have  had background staining on all slides. I have also 3% normal goat serum for blocking.
Any suggestions?? Help!!!
Ron Martin
-----Original Message-----
From: Rob Crawford []
Sent: Monday, March 10, 2003 4:31 PM
To: Martin, Ronald
Subject: RE: beta galactosidase immunos

Hi Ron-
	Couple of questions:
	1. How is the tissue prepped?
	2. Who's primary are you using?
	3. What is your secondary(tertiary, etc...)
	4. What tissue? 
	If you are using an ABC method, a likely culprit is endogenous
biotin.  There are commercial kits available to solve this problem or
you can make up the solutions yourself. I recommend the kits, but if you
want the info on the do-it-yourself, let me know.  
	Watch out for monoclonal antibodies as the secondary will
recognize endogenous Ig and Fc receptors.  You can block these but if
you can go w/ a polyclonal in mouse it's much easier.  
	I may have other info based on the answers to the above.
	Hope this helps.

Rob Crawford

Univ. of Washington
Chamberlain Lab

-----Original Message-----
From: Martin, Ronald [] 
Sent: Monday, March 10, 2003 12:58 PM
Subject: beta galactosidase immunos

Fellow netters,
I am attempting beta gal immunos on mouse tissue and getting a lot of
background stain. I have tried 3% H202 as is and made up from 30% H2O2
in methanol. I have also tried different antibody titration's from 1:200
up to 1:4000. I am redoing the 1:2000 and 1:3000 tomorrow but need some
suggestions on how to reduce the background staining. I also use the
recommended 3% goat serum.
Ron Martin

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