[Histonet] Re: Efficacity of fluorochrome in Immunofluorescence

Andrea Marion amario3 <@t> uic.edu
Tue Jan 24 14:29:06 CST 2012

Hi Natalia,

What type of tissue are you working with, and how was it processed? Are
these paraffin sections or cryosections? What fixative did you use? These
details can help narrow down a potential problem. If you post your
protocol that will help.

You might be seeing autofluorescence from the tissue itself, especially if
you are working with paraffin sections or formaldehyde-fixed samples. See
what the tissue looks like with no secondary antibodies - if you see the
same fluorescence, you have autofluorescence coming from the tissue
itself. You will need to either process the tissue differently, try to
block the autofluorescence, or amplify the signal of your antibody
staining above the level of the autofluorescence (using avidin/biotin or
TSA for example).

To check for nonspecific binding of the primary or secondary, you can run
a panel of stainings, leaving out the primary antibody or replacing with a
general isotype control, as Kim suggested.

Andrea Marion
Graduate Student
University of Illinois at Chicago

Hi everybody!

Maybe you can help me.

I'm trying to do immunofluorescences to see the colocalisation between two
proteins (PSD + Oligomeres) using Oregon Green (goat anti mouse) and Texas
Red (goat anti rabbit).
But my probleme, is that the analysis of the slides reveals too much
colocalisation. I mean, all seems yellow, as if both fluorochromes show
the same thing.

It's the first time I do fluorescence, so I don't know where could come
the problem.
I tried to do a simple staining, 1 slide with my first antibody and its
fluorochrome, and the second slide with the other first antibody with its
fluorochome and then check the result with the filter red and green. But
it's strange because I see both color as if I had put both fluorochromes
(second antibodies).

Has somebody already had this problem?
What can I do to resolve it?

Thank you very much for your help.

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