no background on the negatives

-----Original Message-----
From: Starost, Matthew (NIH/OD/ORS) [mailto:starostm@ors.od.nih.gov]
Sent: Tuesday, March 11, 2003 8:30 AM
To: Martin, Ronald
Subject: RE: beta galactosidase immunos


What are your negative slides looking like - do you get the same background
when you remove the primary antibody?

Matthew F. Starost DVM, PhD
Diplomate, ACVP
NIH, Bldg. 28A, Rm. 115
9000 Rockville Pike
Bethesda, MD 20892
Phone: 301-496-4465
Fax: 301-402-1068
E-mail: starostm@mail.nih.gov


-----Original Message-----
From: Martin, Ronald [mailto:Ronald.Martin@umassmed.edu]
Sent: Tuesday, March 11, 2003 8:17 AM
To: Rob Crawford
Cc: histonet@pathology.swmed.edu
Subject: RE: beta galactosidase immunos


Rob,
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!!!
Thanks,
Ron Martin
-----Original Message-----
From: Rob Crawford [mailto:rcthree@u.washington.edu]
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
rcthree@u.washington.edu

Univ. of Washington
Chamberlain Lab
206-221-6386(phone)
206-616-8272(fax)


-----Original Message-----
From: Martin, Ronald [mailto:Ronald.Martin@umassmed.edu] 
Sent: Monday, March 10, 2003 12:58 PM
To: histonet@pathology.swmed.edu
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.
Thanks,
Ron Martin