Looking up from her microscope, Judy Trogadis <[log in to unmask]>
was heard to say:

> 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,

I have heard of others experiencing similar problems using avidin-biotin.
What are you using as your blocking reagent?  If it contains egg-albumen,
that could well be the source of your background, as egg-white is very
rich in biotin.  I would also  suggest an intermediate blocking
step (between the primary and secondary antibodies) using normal serum
from the host animal your secondaries are derrived from (ie, if you're
using biotinylated-goat-anti-rabbit as a secondary, block in 5% normal
goat serum with 0.5% tween-20 for an hour or so before you apply the
secondary).

If you find something that works, let us know...I'm always collecting
immunostaining tricks for my toolbox.

Cheers