Worm Breeder's Gazette 5(2): 45
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
Making use of our laser microbeam we have developed a method for fixing eggs with glutaraldehyde at room temperature. This fixation preserves nicely cell junctions, cytoskeletal components, including microtubuli, and other ultrastructural markers. Thus it complements the hot osmium procedure, useful for membrane visualization . Single- egg embedding at any desired stage can be reliably done, allowing EM series to be made from any embryo which has been video-taped in vivo. The egg is mounted in the extrusion medium detailed elsewhere ( Laufer and von Ehrenstein, this WBG), containing 0.3% glutaraldehyde. To allow penetration of the glutaraldehyde, the eggshell is punctured with a single laser shot at the desired developmental stage. Cytoplasmic streaming and movement of yolk granules cease within a few seconds, showing that fixation starts immediately. Fixation is for 1 hr at room temperature. Additional landmark scars can be made in the egg by laser shots, facilitating orientation during embedding and sectioning. To facilitate handling, the egg is placed into a slab of 4% agar, then washed for 15 min with 0.15 M sodium cacodylate buffer at pH 7.4, and post-fixed at 4 C for 3 hr with 2% osmium tetroxide in 0.1 M sodium cacodylate pH 7.4 followed by three 10 min washes with the same buffer. Dehydration and embedding are as described previously. Without glutaraldehyde, the hole in the eggshell apparently reseals and embryos develop normally (see, Laufer and von Ehrenstein, this WBG) , if care is taken to puncture the eggshell in a location where it is not in contact with the embryo. Otherwise the heat generated by the laser shot lyses the embryo.