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

[C.M. van der Loos]

   Hello Swaram,

   I have been testing this Jackon protocol once and could not figure out
   a good staining either. I presume that this type of protocol only
   works under "ideal" conditions. That means if you have exactly
   titrated all your reagents perfectly. Just a little too much of one
   reagent will result into non-specific, or better to say "unwanted"
   staining.

   As mentioned on Histonet, the approach to biotinylate one primary via
   the Dako ARKit (or even both primaries via the Zenon kit), is much
   more safer.

   Good luck,

   Chris van der Loos, PhD
   Dept. of Pathology
   Academical Medical Center M2-230
   Meibergdreef 9
   NL-1105 AZ Amsterdam
   The Netherlands

   phone:  +31 20 5665631
   fax:    +31 20 6960389
   e-mail: [1]c.m.vanderloos <@t> amc.uva.nl


   ----- Original Message -----
      From  Swaram <swaram <@t> myrealbox.com>
      Date  Thu, 03 Feb 2005 10:59:32 -0800
        To  HISTONET <histonet <@t> lists.utsouthwestern.edu>
        Cc  histonet <@t> 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
    ! ;  t

References

   1. mailto:c.m.vanderloos <@t> amc.uva.nl