With this method, you need to use different species of primary, like one mouse and one rabbit primary. You can actually use two of the same species if you put down one primary, followed by secondary, link and substrate, THEN the second primary,secondary, link and substrate.  You may or may not use different substrate systems, like peroxidase and alk. phos.  I've just recently begun to play around with immunofluorescence, and using two different fluorescently labeled secondary's to visualize two antigens, using Vector's fluorescent secondary antibody kits (500 ul each of AMCA, fluorescein, and TexasRed labeled secondary antibodies).  It's fun and great looking when you get it to work, and impressive.
Joyce Kotzuk, UNM pathology dept.

>>> Dana Settembre <settembr@UMDNJ.EDU> 03/24/00 07:02AM >>>
On Thu, 23 Mar 2000 cklein@mail.mdanderson.org 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
USA