RE: Method for double immuno staining

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From:"Bennett, Catherine (Katie)" <>
To:"" <>, "''" <>
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Vector Labs has a fairly good web page describing the basics of multiple
labeling IHC protocols.  It's a good place to start.  Go to  and look under the Immunostaining heading.

You are correct that the staining is basically a layering of the two stains
on top of one another.  But be sure your first stain is with something
really permanent like DAB.  I would recommend using different enzyme
substrates (peroxidase and alk phos) for each of the antibodies you are
staining with.  As to doing two different antigen retrievals - that may be
needed, depending on each antibody.  Be prepared to do some troubleshooting.

Catherine "Katie" Bresee Bennett
Sr. Technical Associate
Lovelace Respiratory Research Institute
Albuquerque, New Mexico

> ----------
> From:
> Sent: 	Thursday, March 23, 2000 1:19 PM
> To:
> Subject: 	Method for double immuno staining
> 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.

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