Hoescht 33342 vital staining.

Gunnar Kleemann kleemann at cricket.unl.edu
Fri Apr 7 14:21:29 EST 2000


I was wondering if anyone would be willing to offer me some advice on
vital staining of C. elegans. I have been attempting to make this
procedure work for a number of months with no luck.

I am using the fluorescent stain Hoechest 33342. the stain needs to
penetrate the tissue of the worm and stain the sperm within male worms.
I have soaked worms in approximately 2 and 4 milligrams of stain/
milliliter of M9 buffer for up to 3 hours. While this staining procedure
does not kill the worms (as found for Hoechest 33358) it seems to only
stain the digestive system.,

Additionally I am having trouble distinguishing stained tissue from the
autoflourescent glow emitted by older worms. I think that if the worms
are properly stained than any autoflourescence will be pale in
comparison, but I am mot sure.

The scope I am using may be part of the problem. This stain has a peak
excitement at 350nm and sows peak emission at 450nm, while my scope can
only excite as low as 400nm ,and the dichroic mirror reflects only as
low as 450-700nm

Any suggestions would be greatly appreciated.
Thank you
Gunnar Kleemann
University of Nebraska - Lincoln

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