Jacqueline, for your non-specific background problem I suggest one of the
following:

1) Adsorb the nonspecific antibody with liver acetone powder (any mammal,
available from Sigma and others) to the diluted antibody. Add the powder
(about one-tenth the volume of the antisera), mix well and let sit
overnight. Then spin down to remove the powder, decant and use the antibody.
It should adsorb non-specific antibodies.

2) Use a primary diluent that includes human sera to trap non-specific
antibodies.

3) boost the percentage of animal sera in the diluent - up to 20 percent if
necessary.

Tim Morken
Lab Vision / NeoMarkers
www.labvision.com


-----Original Message-----
From: Jacqueline Miller [mailto:Jacqueline.Miller@UTSouthwestern.edu] 
Sent: Friday, September 19, 2003 10:12 AM
To: gcallis@montana.edu
Cc: histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] background problem

The following is information about a high background problem when using
RatIgG1 control antibodies on sections of human placental tissue:

The primary Ab we are using is a rat anti-human.  We dilute the primary
to get 4 ug/ml.  We dilute the RatIgG1 control Ab to get the same
working concentration.  We were diluting all the antibodies in 0.01M PBS
(pH 7.2-7.4), but switched to a 1%BSA in 0.01M PBS solution in an
attempt to correct the background problem.  The primary is on for 60'
RT.

We have had the background problem with two different Rat IgG1 controls
from two different companies.  The one we are using now is much worse.

The secondary is a biotinylated donkey anti-rat IgG F(ab')2 without
cross reactivity, and is on for 30'RT.  However, we have done several
test runs substituting PBS for the primary, and there is no staining. 
We also used a MsIgG1 control antibody on the same type of tissue with a
Vectastain MsIgG1 ABC kit (serum, secondary, and ABC), and had no
staining.  

We wash the slides in PBS after every step except for the serum block
(before the primary).  We've tried increasing the time of the endogenous
block (after the secondary) from 5 min. to 30 min. and decreasing the
time of the ABC (avidin and biotinylated horseradish peroxidase complex)
from 45' to 30'.  We stained the paraffin sections using the citrate
antigen retrieval method, once with a trypsin treatment, and once with
no treatment at all.  In all the treatments/no treatment, all the slides
with the RatIgG1 control stained dark red, and all the slides with only
PBS for the primary did not stain at all.  

Just a couple of days ago, we stained some frozen sections of the same
type of tissue, as well as some sections of an older tissue, and once
again, the sections with the RatIgG1 stained and the ones with only PBS
did not stain.

Any advice or suggestions from anyone on how to solve this background
problem would be greatly appreciated.

Thanks in advance,

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


>>> Gayle Callis  09/15/03 04:20PM >>>
Did you do a dilution panel at beginning to determine that 4 ug/ml is
the
correct optimal working concentration?  4 ug/ml could be too high a
concentration, depending on what you are staining for, and some kit
secondaries may also be too concentrated.  You did not say exactly how
you
were doing the staining and what for??  A kit, Strepavidin, etc, etc.
specific secondary, ???  F(ab')2 secondary??? A few more specifics help
as
there are too many sources of background lurking to shoot us down! 




Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology 
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)



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