[Histonet] Background with DAB staining

Greg Dobbin dobbin <@t> upei.ca
Thu Oct 7 08:01:59 CDT 2004


Sotiris,
Have you considered blocking for endogenous biotin?
Greg


Date sent:      	Thu, 7 Oct 2004 13:25:03 +0100
From:           	"Mandy Townsend" <Mandy <@t> serotec.co.uk>
To:             	"Sotiris Tsalavos" <tsalavos <@t> fleming.gr>,
	<histonet <@t> lists.utsouthwestern.edu>
Copies to:      	Subject:        	RE: [Histonet] Background with DAB staining

> 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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~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
Greg Dobbin
Pathology Lab
Atlantic Veterinary College, U.P.E.I.
550 University Ave.
Charlottetown, P.E.I.
Canada,  C1A 4P3
Phone: (902)566-0744
Fax: (902)566-0851
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
Happiness is a journey, not a destination.




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