Re: Method for double immuno staining

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From:Dana Settembre <settembr@UMDNJ.EDU>
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On Thu, 23 Mar 2000 wrote:

> A doctor has asked our lab to do a double immuno stain using both p21 and ki 67.
> My question is---where can I look for a relatively easy to understand method to
> accomplish this?  I suspect that I will need to use 2 different chromagens(dab
> and aec) but do I have to retreive the antigens twice, block peroxidase twice,
> etc?
> My assumption is that I should proceed with one antibody stain form start to
> finish and then for the second antibody I should start the procedure from the
> point of primary antibody application to the second chromagen.
> Any help anybody can give regarding either the method or where to look up the
> method would be greatly appreciated.
> Sincerely,
> Christine Klein
> MD Anderson Cancer Center
> Houston, Tx.
Hi Christine,
I have done double staining and triple staining just a handful of times
but it works.  I use the Dako stainer but it can be done by hand too.

1. Pretreat once
2. Block endogenous peroxidases once
3. Add the "first" primary antibody for your usual time, rinse
4. Add the "second" primary antibody for the usual time, rinse and 
5. Detect with the two different systems. 
Dana Settembre
Immunohistochemistry Lab
Pathology Department
University Hospital
Newark,  New Jersey

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