Fixative for Fruitfly larvae

[waalkesmr]

Hi,
I am conducting an experiment and trying to fix fruit fly larvae. My goal is to image them using fluorescence. The fruit fly's genotype is pcnaGFP, which means they have green fluorescent protein highlighting neural stem cell proliferation. So I need is a fixative that kills the larvae, does not effect the green fluorescent protein structure, and isn't fluorescent itself. Let me know if you have any ideas. Thanks!
-Matthew

[Hobbyst46]

Hi,
I am conducting an experiment and trying to fix fruit fly larvae. My goal is to image them using fluorescence. The fruit fly's genotype is pcnaGFP, which means they have green fluorescent protein highlighting neural stem cell proliferation. So I need is a fixative that kills the larvae, does not effect the green fluorescent protein structure, and isn't fluorescent itself. Let me know if you have any ideas. Thanks!
-Matthew

I know nothing about mounting or fixing larva. But there is a mounting medium named Fluoromount, or Floromount, which can be used for mounting directly from water. And has particularly low auto-fluorescence. Perhaps it will kill the larva as well ? I would check its properties and use in the literature.

[MicroBob]

Hi Matthew,
these are quite a few requirements!
According the mounting media: Entellan Neu and Histikitt are said to have no autofluorescence. But I don't know how well they preserve the autofluorescence in your larvae. Does your preparation have to conserve the larvae for long term storage?
A common mountant for shorter storage times is Hoyers mix, but I can't tell you whether it has autofluorescence and whether it l leaves the autofluorescence in your larvae intact.
With which wavelength do you exite?
Enclosed you can find the autofluorescence of some mountants excited with blue light.

Bob

[waalkesmr]

Thanks for the replies! I will check out some of the fixatives you mentioned. Bob, to answer your questions, the wavelength we expose is at 488nm and we only need the slides for a very brief period of time (I hope to image the larvae immediately after they stop moving, the quicker the better).

Since what fluoresces is a protein, would an oxidative agent be a good starting point for a fixative? Also (I'm an amateur at this right now) which one of the mountants that you show has the least auto-fluorescents? Funnily enough, we use transparent Elmer's glue and it works well. But our protocol right now is that we mount the larvae live and we use nothing to kill them. This is fine for our purposes except they might still be moving even 1 hour after mounting. Our ultimate goal will be to increase the rate at which we image these animals and thus may involve imaging them in the fixative and forgoing mounting.
-Matthew

[MicroBob]

Hi Matthew,
what i proposed are mountants to produce permanent slides. The ones with the blackest background show the least autofluorescence. These media don't fix the specimen apart from Hoyers mix.

Since you only need the larvae for a short time I would first kill the larvae (cocaine, nicotine, alcohol, strong pain killer?) You will have to test with what you can get.
Then try mounting in glycerol that shows no autofluorescence and see whether you larvae's autofluorescence keeps going for long enough.

Bob