Molecular oxygen is a major contributor to photobleaching. Have a look at this article http://wwwuser.gwdg.de/~uboehm/images/39.pdf and make use of the Molecular Probes Handbook--here are some suggestions (https://www.thermofisher.com/us/en/home/references/molecular-probes-the-handbook/tables/scavengers-of-reactive-oxygen-species.html).

Many thanks Matthew!! will check them both!!

Dear Elah

The following article discusses different anti-fading mounting media in detail.

"Quantitative Comparison of Anti-Fading Mounting Media

for Confocal Laser Scanning Microscopy"

M. Ono, T. Murakami, A. Kudo, M. Isshiki, H. Sawada, and A. Segawa

J Histochem Cytochem, 49:305–311, 2001

In my work I had good experience with the antifading mounting media from Molecular Probes; Eugene, OR. USA

Best regards,

Daniela

Here at Molecular Probes we have a range of different antifade options (such as ProLong Diamond), which I have helped develop. However, none of them have been tested with Nile Red, nor have I heard of other companies testing theirs for that dye, so we can't say for certain about that dye. Feel free to call us in Tech Support if you would like some guidance (541-335-0353).

Many thanks Daniela and Jason. Maybe you have another suggestion for another triglycerides dye ?

Well, there are some BODIPY dyes that can be used as nonpolar and amphiphilic probes, such as BODIPY 493/503 (catalog D3922 from us at Thermo Fisher Scientific), but they aren't necessarily better for photostability. Same goes for dodecanoylaminofluorescein (D109). However, BODIPY dyes have been shown to be protected by ProLong Diamond, and fluorescein and its derivatives are protected by all of our antifades. So perhaps go with one of those and ProLong Diamond (which hardens for archiving or later viewing) or SlowFade Diamond (which stays liquidy for immediate viewing)?

Hi!

I was just wondering could this be due to the lipophilic dye not bleaching but kind of getting absorbed to the mountant. Maybe try mounting in a aqueous solution (PBS or similar) just to check wether that might help. Also, in general, lipophilic dyes are probably best viewed quite fresh?

Veijo, there is precedent for that. The issue is that most aqueous-based mounting media have at least some percentage of glycerol in them (for coverslipping and preventing outgassing bubble formation under the coverslip), and the glycerol can extract lipophilic dyes from samples. But it doesn't seem to happen to all such dyes with all such mountants. For instance, I've labeled cryosections with DiI C18 and mounted in ProLong Gold and stored for weeks with no apparent extraction.