Mounting with PBS for Microscopy - (Jan/22/2006 )

Greetings,
I've had some issues in the past mounting with Mowiol. It seems after time that the cells lose some structure. I'm particularly interested in ruffles and so on. If I do my microscopy as they have just finished drying with Mowiol, the cells look great. 3-4 days later at storage in -20, the cells don't look so nice, and 2 weeks later they are virtually unusable.

I decided then to go to a PBS based mounting medium, and seal with nailpolish. Yesterday I tried this, but all my cells are "dry" today, and only exhibit autofluorescence signal. I made a 50/50 PBS glycerol mixture, loaded about 3 microliters onto the slide, then mounted the cover slip. Immediately I tried to seal it with nail polish.

Anyone have any suggestions?

Maybe you can try Fixogum to seal the edges of the coverslip. I have no clue if it works though cause I've always used glycerol-based mounting medium.

Firstly, never use nail polish with fluorescence slides as the isopropanol in it leaches out into the mounting medium quenching true fluorescence and giving heinous autofluorescence.

Invest some money in a worthwhile anti-fade mounting medium. Do not make your own. Molecular Probes sells one called ProLong Gold that dries hard. It is AMAZING. I believe it also comes with DAPI if desired although I haven't used that one so cannot comment directly.

There are other good ones too so if you are interested in more information contact me directly.

I guess my problem with the "hard" polymerizing based mounting agents is that they "crush" the cells a tiny bit. Well...actually its more than a tiny bit.

I'm looking at FRET in the lifetime domain between two fluorophores. If I prepare the samples fresh, I get a nice FRET signal. If I let it stay for more than 24 hours, this is reduced. After a couple days it is totally gone. The majority of the FRET occurs in the membrane ruffles. These structures are only clearly visible before the mowiol/PVA hardens completely.

So, if I wanted something that didn't harden, is their a suitable embedding medium or am I out of luck?

Hi,

i use Fluorescent Mounting Medium from DakoCytomation
it contains 20% glycérol and an anti-fading agent...
it's no necessary to seal the coverslip
i've stored the slides at 4C for a long time

Sébastien_