[Histonet] RE: immunofluorescence amplification...
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].
Chris van der Loos, PhD
Dept. of Pathology
Academic Medical Center M2-230
NL-1105 AZ Amsterdam
Date: Mon, 15 May 2006 16:44:28 -0700 (PDT)
From: Adam Perry
Subject: [Histonet] immunofluorescence amplification...
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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