3 Electron Microscopy Techniques
3.2 Experimental EM Methods
3.2.4 High pressure freezing/freeze-substitution of C. elegans embryos and L1 worms
Embryos at the 3-fold stage of development and L1 worms less than 6 hours old were frozen with a Bal-Tec HPM 010 high pressure freezer. Freezing of worms was optimized by using slot and Chien EM grids as spacers between the flat surfaces of 2 Bal-Tec specimen holders. Best freezing was achieved when the thickness of the spacer(s) closely matched the diameter of the particular stage of the worm. The frozen samples were then substituted using a picnic cooler and dry ice or a Leica AFS device using the following protocol:
Substitution cocktail: 1% OsO 4 , 0.2% uranyl acetate in 95% acetone:5% methanol
1. 3 days at -80 deg C (dry ice) or -90 deg C (Leica AFS).
2. 12 hours at -25 deg C.
3. 3-4 hours at 4 deg C.
4. 1-2 hours at room temperature.
5. Rinse with dry acetone followed by propylene oxide.
6. Infiltrate and embed in Eponate 12 resin (1A:1B).
50 nm serial sections were stained with 7.5% uranyl acetate for 15 minutes followed by Sato's lead for 3 minutes.
|3-FOLD EMBRYO (low magnification)||3-FOLD EMBRYO( Nerve Ring)||3-FOLD EMBRYO (Amphid Cilia)|
|L1 Larva (Pharynx)||L1 Larva (ALM)||L1 Larva (PLM)|
Richard Fetter, Ph.D.
Dept. of Anatomy/HHMI
University of California, San Francisco
513 Parnassus Ave, Box 0452
San Francisco, CA 94143
phone: (415) 502-3923
fax: (415) 476-3493