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

[Sharon Cooperman]

Hi,

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.

Sharon



>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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-- 
Sharon Cooperman       	     <scoop <@t> mail.nih.gov>
NIH, NICHD, CBMB                     301.435-8417
Building 18T, room 101               301.402-0078 fax
Bethesda, MD 20892