[Histonet] immunofluorescence amplification...

From:Adam Perry

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 microscope and filters are fine)...I was thinking that maybe my antigen of interest is expressed at low levels, so maybe the amplification with the avidin-biotin procedure is important...are there similar ways to amplify fluorecent signal?  I know I can use fluorescent-avidin molecules to label a biotinylated secondary...does this amplify the signal more than a fluorescent labeled secondary alone?

Any thoughts or comments would be great...if you need more details about my specific protocol, please ask...

thanks in advance,

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