[Histonet] RE: immunofluorescence amplification...
I find using a biotinylated secondary and Streptavidin labeled with
fluorophore also works well for amplication, certainly a step up from
directly labeled secondaries where amplification is desired.
At 8:56 AM +0200 5/17/06, C.M. van der Loos wrote:
> Date: Mon, 15 May 2006 16:44:28 -0700 (PDT)
> From: Adam Perry
> Subject: [Histonet] immunofluorescence amplification...
> To: firstname.lastname@example.org
> 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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