Swaram,
What results are you getting?

Assuming that you have worked out each antibody individually first on your
tissue why not try Zenon (Molecular Probes) primary antibody labeling using
different Alexafluors? You can skip all those blocking headaches.

Brett

Brett M. Connolly, Ph.D.
Merck & Co., Inc.
MRL, Imaging Research
WP26A-3000
PO Box 4
West Point, PA 19486
PH 215-652-2501
fax. 215-652-2075
e-mail. brett_connolly@merck.com



-----Original Message-----
From: histonet-bounces@lists.utsouthwestern.edu
[mailto:histonet-bounces@lists.utsouthwestern.edu] On Behalf Of Swaram
Sent: Thursday, February 03, 2005 2:00 PM
To: HISTONET
Cc: histonet@pathology.swmed.edu
Subject: [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.
   [1]http://www.jacksonimmuno.com/technical/examplec.asp
   Please help... !
   Thank you all..
   SWARAM

References

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