[Histonet] Re: antibody elution solution

From:"C.M. van der Loos"

I fully agree with James that 0.1M glycine buffer pH2.0 may work well for eluting bound antibodies from a tissue specimen for double staining purposes. Indeed the removal of the first antibody is hardly complete, not to say isn't working at all! Using glycine-HCl buffer you may remove your second step reagent from a tissue section, but especially high affinity antibodies just stay nicely bound at the antigen in the tissue section. Problem is that the affinity characteristic of a primary antibody isn't mentioned in data sheets! Long time ago investigators tried many ways to get rid of the first set of antibodies and leaving the enzymatic reaction product. Lots of chemicals were tested including oxidation of antibodies and even a kind of electrophoresis system. All procedures shared the problem of high affinity primaries that stayed on the tissue section. There is some older literature available on this point. The only way I came across for excellent removal of antibodies is a
 short heat-induced antigen retrieval method using citrate6.0 (Lan et al. JHC 43:97-102, 1985). Indeed not a single antibody will survive cooking! Obviously this can be done for FFPE sections only, not for cryo's! 
The story above doesn't say that sequential double staining isn't working at all. When applying DAB in the first staining sequence the reaction product shelters the first set of immunoreagents quite effectively. The second primary and second set of detection reagents can no longer cross-react with the first immunostaining sequence. This characteristic makes the DAB reaction product suitable for at least double staining two different cell types.  
However, if you wish to look at mixed-colors marking co-localization of two antigens at one cell, the sequential double staining using DAB in the first sequence is not safe. In this case you need to consider other double staining systems!

Chris van der Loos, PhD
Dept. of Pathology
Academical Medical Center
Amsterdam - The Netherlands

----- Original Message ----- 
>From  jincan  
Date  Fri, 20 Feb 2004 21:04:47 -0800 
To  linda Ross  
Cc  Histonet@lists.utsouthwestern.edu 
Subject  Re: [Histonet] antibody elution solution 
Hi Linda,
Any solution of low pH will work to some extend. The principle is to elute 
first primary antibody (and thus also the secondary antibodies attached to 
it) from sections. Many commercial kits use 0.1N HCL. However, I found 0.1M 
glycine buffer, pH2.0 worked better (5-10 minutes each, two times). The 
buffered solution is milder to the tissue (in preserving morphology) and 
will not reveal as many new epitopes or background as 0.1N HCL. 
Nevertheless, removal of first antibody is hardly complete but only to 
reduce it to a level that would allow differentiation.
Good luck!
James Guo
Lab Manager
ImmunoVision Technologies, Co.
100 North Hill Drive, #32
Brisbane, CA 94005
Tel: 650-302-4622
Fax: 650-558-9621
Website: www.immunovisiontech.com

At 10:21 AM 2/20/2004 -0800, you wrote:
>Does anyone know if there is a commercially available reagent to elute an 
>antibody complex for a doublestaining procedure?  I am trying to double 
>label two mouse primarys using a MOM kit.  I know that Dako includes a 
>doublestain block in a kit but it is not available by itself.  I also 
>called zymed  and inquired about their doublestain enhancer. The tech rep 
>said that this product does not remove the complex and could not tell me 
>how it works....just that it might help me.  Has anyone used this product 
>before?  Thank you....Linda

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