[Histonet] Same Host Ab(fluorescence IHC) HELP...!!!


   Dear Histonetters,
   Here  is the problem that is giving me a lot of grey hairs even though
   I  am only 26. Its making me crazy and I am in deep despair. I cant go
   on like this.. Please help..! :'(
   I  have  to  do  Fluorescent based IHC on two proteins on old paraffin
   fixed  skin tissue. The first antibody telomerase is from Mouse. I use
   goat antimouse Fab2 fragments of Alexa488 for the secondary detection.
   The  second  Ab  that  I have to stain is melanoma cells and I use Pan
   Melanoma  cocktail  from  Biocare  Medicals  for this purpose. This is
   again from Mouse and has several IgG subtypes like IgG1K, IgG2a, IgG2b
   etc.  I use Goat antimouse TRITC as my secondary for this step. Before
   the  application of my second Primary(Pan Melanoma), I use Mouse (H+L)
   and incubate for 30 minutes, and then add (Mouse Fab(H+L) for blocking
   any  epitopes  on  the first primary Ab (telomerase) so that my second
   secondary (TRITC) will not bind to that. However, I have been far from
   successful.  I  have  run out of ideas. There are no good Ab for using
   against  Melanoma  (especially basal melanocytes) from any other hosts
   that work well.
   Here is a scheme that I use from Jacksonlab website.
   Please help... !
   Thank you all..


   1. http://www.jacksonimmuno.com/technical/examplec.asp
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