We use Alexa Fluorchromes with just PBS in the final steps.  We mount in
Vectashield (with or without DAPI) from Vector Laboratories. When stored in
the dark in the refrigerator, fluorescence is still present for over a year.

Kelvin So wrote:

> Dear friends,
>
> Thanks a lot for the quick response on the subject.
> I think I might not have explained my questions too clearly. My apology!
>
> By multiple immuno-labelling, I meant co-localizing the three markers
> concerned in the same frozen sections. Therefore, those multi-chamber
> slides are not helping.
>
> Instead of using Streptavidin Alexa fluorochromes, we are using isotype
> -specific secondaries following the application of the primary
> antibodies. This is at the researcher's request, to avoid any
> complication arising from endogenous biotin reactivity that is supposed
> to be more of a problem in frozen sections.
>
> My question about stability of the fluorescence is in the final slide
> preparation after all stainings are done. We have tried several
> commercial mounting media designed for fluorescence and none of them
> appeared to be satisfactory. Instead of coverslipping,
> we now manage to obtain good enough images with 20x or lower objectives.
>
> I prefer to have some tween-20 in my PBS. This helps to spread my
> reagents easily and more homogeneously over the sections; keep the
> timings more consistent between slides and lessen the risk of drying-out
> artifacts when a big batch of slides is being handled.
> Feedback from the company (Molecular Probes) 'thinks' that the tween is
> OK with the Alexa Fluors and my test run has proven that too. My
> question is : Will the trace of tween-20 that might still be present in
> the DRY stained sections speed up with the fading of fluorescence on
> storage? Because if I am doing a big batch of slides at one time, it
> will take days for the person to finish with the imaging.
> My slides from the test run will serve to prove this later. But I was
> hoping to get a quick answer because the researcher wants this project
> to be done soon!
>
> Any more feedback from experts in the Histoland will be very much
> appreciated.
>
> Kelvin So
> University Health Network
> Toronto, Canada.