[Histonet] Background with DAB staining

[Mandy Townsend]

Sounds like your secondary is reacting with your tissue, unless it has been claimed that is has been adsorbed against mouse.  therefore you should try diluting the secondary in mouse serum to remove any cross reactivity, then hopefully your sections with just secondary will come out negative.

mandy

-----Original Message-----
From: Sotiris Tsalavos [mailto:tsalavos <@t> fleming.gr]
Sent: Thursday, October 07, 2004 1:09 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Background with DAB staining


Hallo.

I'm currently trying out to stain murine spleen and thymous for
dendritic cells using the hamster anti-mouseCD11c antibody.

As a secondary Ab I use biotin goat anti-hamster IgG and then
streptavidin HRP for DAB staining. I'm getting a lot of background in
both types of tissues even in the negative control. Below I have the
protocol that I use. 

 

Frozen tissues cut at 10um placed on gelatin embedded slides.

Air dry for 30min, fix in cold acetone for 10min and then in PBS/0.3%
H2O2 for 10min for the endogenous peroxidase.

Slides blocked with PBS/5% Goat serum for 30min.

Primary Ab anti-mouse CD11c at 1:100 dilution for 1 hour.

Secondary anti-hamster IgG at 1:800 dilution for 1 hour.

Streptavidin HRP at 1:500 for 30min.

Use DAB prepared solution 0.05% DAB- 0.015% H2O2 in 0.01M PBS pH 6.8

 

The DAB I use is from Sigma. 

I'm not sure that the background is still from the endogenous
peroxidases in the tissues or something else.

I have tried staining the tissues with just the secondary Ab from 1:200
up to 1:3200 dilution. In the high 1:3200 get very little background
however I'm afraid that it might be too dilute to bind the primary Ab.

 

Any suggestions would be greatly appreciated. Thanks

 

Sotiris Tsalavos

Institute of Immunology

B.S.R.C. Alexander Fleming

Athens, Greece

 

tsalavos <@t> fleming,gr

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