[Histonet] background problem

[Jacqueline Miller]

Nidal,

Thanks for the advice.  In the past, we've used a 0.5% hydrogen peroxide solution for 5 minutes.  We tried increasing the time, but it didn't help.  We block with 1.5% normal donkey serum for 20 minutes, and our secondary antibody is a donkey anti-rat.  I haven't had a chance to talk to my boss yet about using Tween 20 in the washes.

Jacqueline Miller
Research Asst I
Department of Obstetrics and Gynecology
UTSouthwestern Medical Center at Dallas



>>> NIDAL E MUVARAK <nmuvarak <@t> facstaff.wisc.edu> 09/15/03 03:55PM >>>
Jacqueline,
The background could be due to several factors. If you're doing immunoperoxidase staining, make sure you quench any endogenous peroxidase activity well before adding the substrate-DAB (if applicable). Some people recommend 0.03% hydrogen peroxide for 5-10 min; others do it @ 0.1-1% for 5-10 min. Also, blocking helps reduce background. Blocking works best when the serum used for blocking is derived from the same species in which the secondary antibody is raised. Some people use 1% blocking serum for 10 min. Others use it @ 1-2% for one hour. Last, I add Tween 20 to my washing buffer at 0.3%. Not only does it reduce the background, but it helps break the surface tension of drops when adding a drop of solution to the tissue, which makes the drop spread evenly on the tissue. Hope this helps. Goodluck.

Nidal E Muvarak
Associate Research Specialist
Vascular Tissue Biomechanics Laboratory
Department of Biomedical Engineering
University of Wisconsin-Madison
1550 Engineering Dr.; Rm. 2158
Madison, WI 53706-1609
Lab: (608) 265-8921; Office: (608) 265-4205; 
Home: (608) 256-7934; Cell: (608) 332-6068
http://vtb.bme.wisc.edu 

----- Original Message -----
From: Jacqueline Miller <Jacqueline.Miller <@t> UTSouthwestern.edu>
Date: Monday, September 15, 2003 3:02 pm
Subject: [Histonet] background problem

> Hi,
> 
> We're having trouble with high background when using Rat IgG1 
> antibodies (4 µg/ml) on paraffin-embedded sections of human 
> tissues, with or without retrieval procedures.  Does anyone have 
> any advice?
> 
> Thank you,
> 
> Jacqueline Miller
> Research Asst. I
> OB/GYN Dept.
> University of Texas Southwestern Medical Center
> Dallas, TX
> 
> 
> 
> 
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