[Histonet] ICC Background

[Adam Perry]

Hey Amy,

I'm sure this will be the first of many emails asking for clarification...but here goes:

1) How are you visualizing your antibodies and what is your "background"?  Is it fluorescence and excess autofluorescence?  Is it DAB/NiDAB and excessive non-specific chromogen deposition?  Are you using any amplification systems (avidin-biotin-HRP, tyramine signal amplification, etc.)?

2)  What antibodies are you using?  What species are they raised in and what epitopes are they directed against?  Have you run a titration of your primaries and secondaries...if so, what concentrations have you tried?

3)  Are you using a blocking step?  If so, what are you blocking with?

4) What buffer are you doing rinses/incubations in?  Tris-buffered saline, phosphate-buffered saline, or other?  Is there any detergent in your buffers (Triton X-100, etc.)?

5)  Have you tried an incubation in sodium borohydride to reduce unreacted aldehydes (such as from your fixation) into primary alcohols?

6)  How long are your antibody incubation steps?  And are you doing ICC on free-floating sections or on slides?

I think those are my primary questions for now...

Cheers,
Adam

Adam Perry
Department of Physiology and Biophysics
University of Illinois
Chicago, IL 60612


acjanes <@t> bu.edu wrote: Hi,
I really need some suggestions about the background I am getting when
running immunocytochemistry. I am using mouse brain tissue that is
30microns thick and has been perfused with 4% para. We have used
different primaries and secondaries and we always seem to get really
high background regardless of which primary or secondary we are
using. I have also gotten high background in my non-primary control.
The background is so high I can't even tell if there is any specific
binding. I have run ICC many times in the past and I have gotten it
to work before. I am not sure what is going on so if anyone has any
suggestions please let me know!

Thanks! Amy



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