On Oct. 25, Leon Avery wrote:
>Does anyone know of a way of getting rid of gut autofluorescence?
>Mike Roth and I are trying to stain worms with a fluorescent ceramide
>dye. In cultured mammalian cells this dye gets trapped in the golgi,
>so you can use it to look at membrane trafficking and other neat
>stuff. Mike would like to be able to do similar things with C
>elegans. So we fed worms the dye: they eat it, and we think it goes
>into intestinal cells. Unfortunately, gut autofluorescence in both
>fluorescein and rhodamine channels makes it impossible to be sure, or
>to see where it goes.
>Would Flu mutants be any use? Do the fluorescent granules go away
>when you starve the worms? What the Hell are these granules, anyway?
>Lysosomes, maybe? Some kind of storage granules?
======================
Leon:
The autofluorescent granules in the gut cells are definitely lysosomes,
as George Clokey and I showed in 1986 (The autofluorescent "lipofuscin
granules" in the intestinal cells of C. elegans are secondary lysosomes,
Mech. Aging Develop. 35:79-94).
There is no "treatment" that will rid you of autofluorescence. One
possibility is to use daf-4 mutants, which have much lower
autofluorescence because they are strongly deficient in endocytosis into
the gut cells. If your probe enters by passing through the PM, this might
work. If your probe must be taken up by pinocytosis, it obviously won't
help.
Lew Jacobson 412-624-4647 (Lab)
