Hi Judy,

I'd suggest the following controls to find out what the problem is:
1) repeat the whole procedure but without primary antibody. (Although it is
unlikely that your problem is unspecific binding of the first ab if you
have tried already several different abs)

2) avoid biotin system by using a fluorescently labeled 2nd ab, e.g.
Cy3-conjugated goat anti-mouse. Then there is one step less where
unspecific binding could occur.  If the problem is indead endogenous IgG as
suggested by Ian Gibbins then you also should see a signal with only this
2nd antibody.

3) why do you incubate overnight? Is there a special reason for this?
Although I see no theoretical reason against it, to my knowledge most
people incubate only 1-2 h.

4) Have you checked your tissue for endogenous background fluorescence,
i.e. without any treatment? Some tissues are really nasty in that aspect.

Hope this is of some help,

Steffen



>Fellow immunostainers,
>
>I am using immunostaining protocols to localize several different
>intracellular, some intranuclear proteins. I was hoping to benefit from
>both the ease and high sensitivity of the avidin-biotin system and the
>advantages of recording fluorescent images with a confocal microscope.
>
>The tissue is frozen sections of paraformaldehyde fixed mouse retina.
>Following a blocking step, I used 2 different antibodies, one a mouse
>monoclonal, the other, a rabbit polyclonal. After overnight incubation
>with the primary antibodies, both at R.T. and 4 deg., I applied biotinylated
>mouse/rabbit IgG, followed by fluorescein conjugated streptavidin. To my
>greatest diasppointment, all I saw was a high uniformly fluorescent
>background throughout the entire retina for both antibodies.
>
>Am I leaving out a critical step?
>
>Any suggestions would be highly appreciated.
>
>Judy Trogadis
>Eye Research Institute of Canada and
>University of Toronto
>ph:  416-603-5088
>fax: 416-603-5126
>email: [log in to unmask]


-- -------------------------------------------------------------
Dr. Steffen Dietzel
Zentrum fuer molekulare Biologie Heidelberg (ZMBH)
University of Heidelberg
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Phone: +49/6221/54-6836 or -6879. Fax: -5893
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