Multiple Immmunofluorescence Labelling

From:Kelvin So

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

Kelvin So
University Health Network
Toronto, Canada.

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