Hi all
I am a research student working on C elegans and trying to study muscle aging. I am following the protocol mentioned below :
1 )Wash worms off the plate in water, PBS, or M9.
2) Wash 2 times to remove excess bacteria (spin down at 3000rpm X 1min).
3) Remove as much supernatant as possible.
4) Add 10 mL worms to an eppendorf tube.
5)Freeze tube in liquid nitrogen.
6) Lyophilize worms by spinning in speed vac (~5 minutes).
7) Add 3-4 drops ice cold acetone and put in freezer 3 min.
8) Remove acetone with pipetman (or aspirate carefully) and speedvac to remove
residual acetone.
9) Add 2U rhodamine-phalloidin to an eppendorf tube and speedvac to evaporate
methanol. Resuspend in 20 mL S mix.
10) Resuspend dry worms in S mix/phalloidin.
11) Stain in the dark at RT for at least 30 min.
12) Wash 2X in 1 mL PBBT.
13) Resuspend in ~20 mL PBS.
14) For viewing, mix equal volumes of worms and mounting medium.
15) Mount on an agar pad and examine under the microscope.
My doubts are :
- I want to know that at what magnification should I view the muscles so that they appear very clear ? is there any specific one which is required to be reported ?
- what should be the sample size I should use ? I have previously worked with 15-20 worms, but I could see just 2 or 3 worms.
Thanks !