[Histonet] RE: immunofluorescence amplification...

From:"Andrea T. Hooper"

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.

Good luck!

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: 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
>    now.
>    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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