[Histonet] RE: triple staining

[C.M. van der Loos]

Dear Anjan,Your plans are fine. Don't worry about the following chromogens:DAB / Vector Blue / Liquid Permanent RedThese chromogens easily survive the second HIER procedure without any changes. Take care of the dilutions of the primary antibody after the second HIER: dilutions might be higher as usual (because of two HIER procedures).Lots of success!Chris van der Loos, Amsterdam, Netherlands Date: Fri, 19 Dec 2008 23:49:57 +0530
From: anjan kumar <drvet_anjan <@t> hotmail.com>
Subject: [Histonet] (no subject)
To: histonet <@t> lists.utsouthwestern.edu

hi,
I am Dr. Anjan Kumar working as Junior Research Scientist  at Madras veterinary college, chennai, india. currently  i am pursuing my PhD  in
veterinary pathology and i am working on canine mammary tumours for my
theses work and i have planned to work on triple immunohistochemistry using
CD44, CD24, EPCAM AS ALL these antigens are surface markers of stem like cancer cells and they all are co
localized. i am finding it very difficult to select 3 different
chromogens. i have planned to procure monoclonals from Thermo - Lab
visison i.e 

the following are mouse monoclonals:
CD44: IgG2a

CD24: IgM / �

ESA:  IgG1 / �

i have planned to use 2 detection system i.e alkaline phosphatase and HRP
due to funding constraints i am forced to settle for single goat anti - mouse secondary antibody
currently
im confused with everything and i am struck here
And another doubt ... according to
multiple immunostaining methods by Chris Vander loos after double
immuno histochemistry we can proceed for one more HIER and perform
another single immunohistochemistry so this mounts upto triple
immunohistochemistry right! coming to the point, my doubt was after performing
HIER WILL the chromogen present earlier will be lost along with the
immune complex.
 i have stopped all my work here and dont know how i can proceed...
please help me with your valuable suggestions.
can any one plz suggest me  established triple immunohistochemistry protocol.
regards,
Dr anjan.