=
05/10/2004
These are the responses I got regarding live worm =
immobilization:
1) How about a paralyzed mutant background (e.g. unc-54). You could=20
feed animals on unc-54 dsRNA-expressing HT115. Maybe combined with a=20
moderate dose of phenoxypropanol would do the trick.
2) May be you can use an anesthetic like tricin tetramisole. It gives=20=
a relatively good anesthetic effect up to 30-45 min still allowing=20
ovulation process to continue. In my experience give large quantities=20=
of anesthetic for 5 min or so and dilute it immediately.
3) We immobilized worms for up to about 6 hours successfully using a=20
combo of tricaine and tetramisole (Development. 1999=20
Oct;126(20):4489-98. ). It worked OK but it was hard on the worms- the=20=
rate of growth cone migration was SUBSTANTIALLY reduced in immobilized=20=
worms vs free-roamers.=A0 Those were brutal experiments and in the next=20=
paper when I did growth cone analysis (on an unc strain) I just held my=20=
breath and took pictures and hoped the worms didn't move too much-=A0this=20=
worked OK, so maybe putting your worms in a paralyzed background might=20=
be worth a try.
4) I have done the IFT (movement along cilia) assays using 10 mM=20
levamisole as an anesthetic on (one and the same) adult worm(s) on one=20=
and the same slide, spaced up to 20 minutes apart. It worked. The worms=20=
were still OK the second time around for getting a short (less than one=20=
minute) IFT movie out of them. I had taken them off the microscope=20
stage to give them a chance to recover from the fluorescent light beam=20=
and get "good" GFP signal back for the second "movie session". I know=20=
this is not exactly the time scale that you are looking for, but it=20
might be a start to try to figure out what works for really long time=20
scales.
5) Combining the anesthetic cocktail (0.2% tricaine and 0.02%=20
tetramisole) in a 2% agarose pad. Then use a temperature-controlled=20
stage and flowed water cooled on ice over the coverslip for the=20
duration of the imaging. Managed to get movies of up to 6 hours. The=20
cooling helps to slow movement of the worm (the anesthetics aren't=20
great), and greatly improves viability of the worms. I had huge=20
problems with laser damage and worm death without the cooled stage.
