[Histonet] Same Host Ab(fluorescence IHC) HELP...!!!
swaram <@t> myrealbox.com
Thu Feb 3 12:59:32 CST 2005
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..
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