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 / κ ESA: IgG1 / κ 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. Junior research scientist Department of Veterinary Pathology, Madras veterinary college, chennai, india. Make sure your wardrobe reflects the latest trends and styles in the world of fashion. Try it! _________________________________________________________________ Much more than email - don't miss out on the rest of the Windows LiveT. http://www.microsoft.com/windows/windowslive/events.aspx _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet