[Histonet] Same Host Ab(fluorescence IHC) HELP...!!!
scoop <@t> mail.nih.gov
Thu Feb 3 13:21:01 CST 2005
I have had the same problem in the past with blocking with Fab
fragment. My solution to the problem was to label the primary
antibody and use a secondary antibody directed against that label.
Suggestions for labels for the primary antibody are biotin (kit from
Vector or Molecular Probes - you could use a fluor conjugated
Streptavidin as your secondary) or digoxigenin (kit from Roche). You
could also just try to label the primary with a fluor. I did this
once with a kit from molecular probes that worked well.
Another possibility suggested to me by someone was to incubate the
primary and secondary antibodies together in a tube and then put that
complex on the tissue. You would need to work out the concentration
of each. I never tried this because it was quicker for me to label
the primary Abs.
> 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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Sharon Cooperman <scoop <@t> mail.nih.gov>
NIH, NICHD, CBMB 301.435-8417
Building 18T, room 101 301.402-0078 fax
Bethesda, MD 20892
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