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


   Dear Histonetters,
   I  did  try  the  Alexa  Zenon  labelling  kit for labelling my Second
   Secondary  for  MART-1  (from  Upstate).  But  unfortunately,  even at
   concentrations  of  1:5  of  the  labelling complex, it hardly stained
   anything.  I think, the stearic hinderance is too much for the complex
   to  attach  efficiently  to  epitopes  unmasked  after  citrate buffer
   cooking on the paraffin fixed slides.
   I  am  now  thinking,  if reducing the labelling ratios of (1:3) {that
   Molecular Probes recommends} to 1:1 along with not adding the blocking
   reagent  will  help  me  in  increasing the binding and overall signal
   intensity?  Does  anyone think this is tryable..? even logical? I mean
   if  the  labeled  complx  is  too  big  with  addition  of several Fab
   fragments,  its  reasonable to assume that the complx cant bind to the
   epitope  well. (I find the edge staining effect, which probably is the
   washed  off  Ab  complx). So if I reduce the amount of Zenon labelling
   reagent  (which is Fab fragments bound to Alexa dye) to almost 2-3 per
   primary  Ab, then the complex has better access to the epitope and the
   maze of the paraffin world.. ?
   Thanks  for  the  suggestions  on  direct  labelling  of  the  Ab to a
   fluorochrome,  but I find that to do that.. most of the Abs have to be
   free from Glycine/NaCl buffer and unfortunately mfgrs dont sell them.
   Thanks  to  you  who  responded  and  sparked  my  interests  in these
   solutions.. I wish I can get some more tips..
   Swaram, UCSF
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