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Materials:

disposable scapel	#15 (round tip) Feather brand
subbed slides	0.1 % poly-L-lysine (MW>300,000)
	0.2 % gelatin
	0.01 % chrome alum
	(dip slides in solution and dry overnight)
Whatmann paper	for ease cut into 1 inch square pieces

Procedure:

1. Dissect embryos from gravid hermaphrodites
   • pick gravid hermaphrodites into 100 ul of H20 on a subbed slide
   • cut hermaphrodites near vulva with disposable scapel to release embryos
2. Freeze-fracture embryos
   • remove practically all of liquid from slide with pipet (~90 ul)
   • place coverslip on embryos/worm carcasses
   • wick residual liquid out with Whatmann paper until embryos begin to flatten
   • freeze slide on dry ice >20 minutes
   • quickly flip off coverslip with razor blade
   • immerse slide in fixative
   • process for immunofluorescence

Notes:

• Most problems are due to lack of permeablization of embryos when not enough pressure is applied
• Use younger adults to enrich for earlier embryos
• Multiple cuts with the scapel can extrude earlier embryos or embryos still caught in the uterus (after the initial cut, begin near tip and "push" embryos out like toothpaste from a tube)
• For osmotically sensitive embryos, dissect in 150 mM salt
• If the scapel "pulls" too much liquid when cutting, dry the scapel on a Kim-wipe
• To prevent the coverslip from falling back onto embryos, flick the coverslip off while holding the slide with the coverslip side down
• reference: The Nematode C. elegans (Wood, 1988): Methods (p. 587-606)

compiled by Chad Rappleye

Aroian Lab Protocols