[Histonet] RE: immunofluorescence amplification...

From:"C.M. van der Loos"

   Dear Adam,

   The  best  way  of  of  amplifying  fluorescence  signal  is  using  a
   tyramide-based   detection  system.  There  are  kits  available  from
   Perkin&Elmer  and  Molecular  Probes/Invitrogen. First you detect your
   primary  with  a  HRP-labeled  secondary  and then the HRP activity is
   employed  for the amplification reaction ending up with a fluorochrome
   of   your  choice. Also  fluorescence  double  staining  is  described
   performing the  amplification  reaction sequentially with a peroxidase
   blocking  in  between  [Speel et al. 1997, JHC 45:1439-1446: Sensitive
   multicolor fluorescence in situ hybridization using catalized reporter
   deposition (CARD) amplification].

   Good luck!

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

   Date: Mon, 15 May 2006 16:44:28 -0700 (PDT)
   From: Adam Perry 
   Subject: [Histonet] immunofluorescence amplification...
   To: histonet@pathology.swmed.edu
   What  are people's impressions about different (i.e. the best) ways to
   amplify fluorescent signal from immunocytochemistry?
   I'm  working  with  30  um  rodent  brain  sections.   When  I  use  a
   biotinylated  secondary antibody with avidin-biotin-HRP detection with
   DAB  or  NiDAB  I  get  good cellular staining with a primary antibody
   concentration of 1:1,000.
   I  am  now  trying  to perform double labeling and so have switched to
   fluorescence...and  I  don't  seem  to  be getting any specific signal
   I  have tried increasing the antibody concentration from 1:1,000 up to
   1:100  and  still  don't  see  the  faintest hint of staining (and the
   background just gets really bad).
   I've  used  the fluorescent antibody to detect other primaries (so the
   secondary is working in general and my mi! croscope
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