[Histonet] Re: Can Alk phs be run on peroxidase slides?

[Johnson, Teri]

Dana, the answer is yes, it can work (anything is possible, right?). It
does not have to be destained.

Are you looking for colocalization or does the pathologist expect the
other antibody complex to be localized elsewhere?

Co-localization may be difficult to visualize using DAB and a red or
dark blue AP chromogen.

I recommend that you include two positive controls for the AP-labeled
antibody. On one, do the peroxidase/avidin/biotin/DAB sequence just as
your slide has had (regardless of whether there would be any positive
labeling with the first antibody), and then run both using the second
antibody staining sequence. So, one will have the double stain sequence,
and the other just the single AP stain sequence.  You should be able to
tell then if there has been any diminution of signal due to the previous
labeling technique.

Chris van der Loos has reported that DAB has a sheltering effect on
antigens and may actually help keep the 2nd primary from unwanted
binding.

>From the archives:
"1. Sequential double staining only works because of unique effective 
sheltering of DAB chromogen. As far as I know there are no other 
enzymatic chromogens with this sheltering capacity. Always apply DAB 
after the 1st staining sequence!!!!!"


Finally, make sure your second primary antibody is raised in a different
species than your first primary antibody. There are ways around it, and
we can talk further if that's the case.

Good luck!

Teri Johnson, HT(ASCP)QIHC
Managing Director Histology Facility
Stowers Institute for Medical Research
1000 E. 50th St.
Kansas City, MO 64110



Date: Mon, 24 Jul 2006 12:44:48 -0400
From: "Dana Settembre" <settembr <@t> umdnj.edu>
Subject: [Histonet] Can Alk phs be run on peroxidase slides?
To: <histonet <@t> pathology.swmed.edu>
Message-ID: <s4c4c0e3.071 <@t> smtpnpc.umdnj.edu>
Content-Type: text/plain; charset=US-ASCII

Hi,
One of my pathologists is asking if I can take a slide that has already
been stained with a peroxidase/avidin/biotin/DAB detection system and
re-stain with an Alkaline Phosphotase detection system. Can it work?
Must it be de-stained?

Any info will help.  Thanks,

Dana Settembre
Immunohistochemistry Lab
University Hospital - UMDNJ
Newark, NJ


Teri Johnson, HT(ASCP)QIHC
Managing Director Histology Facility
Stowers Institute for Medical Research
1000 E. 50th St.
Kansas City, MO 64110