Worm Breeder's Gazette 15(4): 15 (October 1, 1998)
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.
Center for C. elegans Anatomy, AECOM, Bronx, NY 10461
EM methods for C. elegans generally require the specimens to be cut open with a razor blade during the primary fixation in order to allow the fixative solution to get past the cuticle. Embryos have been particularly difficult to process, as they are too small to dissect mechanically. Microwave energy somehow enhances penetration of fixatives in many EM protocols (1), and has been shown to be helpful in fixing intact adult C. elegans (2). We have tested a new microwave fixation protocol to improve preservation of uncut animals at all stages for immunoEM, where there is need to achieve uniform processing of hundreds of individual specimens at once. Using a specialized microwave oven (Ted Pella) with adjustable energy levels and programmable pulse regimes, aldehyde fixation is achieved within minutes. To preserve antigenic sites, specimens are chilled during irradiation. A 1 cc sample in a glass multiwell dish is positioned at a microwave "hotspot", bathing the dish in 100 cc of ice. A beaker holds a large water load (600 cc) away from the sample. Microwave energy is set at the 20% or 50% level, with a regime of 2 min ON, 2 min OFF, etc. repeated over total period of ten minutes. The ice load rechills the sample between each microwave cycle, so that the worms never become heated above 10oC. After aldehyde fixation, samples are washed in cold buffer, and embedded into agarose as a dense "pellet" just before the agarose sets (Seaplaque or Sigma type VII agarose; gelling temp is 30oC). After the agarose cures at 4oC overnight, small blocks are cut out, dehydrated and embedded into LR Gold resin. Specimens are placed into gelatin capsules and cured under UV in a Pella cryochamber at -20oC as described previously (3). Thin sections of the pellet are collected onto nickel mesh grids and treated with primary and gold-linked secondary antibodies. Adults, larval stages and embryos are adequately preserved and also become better embedded than without the microwave treatment. We use different buffered aldehyde mixtures, depending upon antigen sensitivity (3). 1. Login GR, Dvorak AM. J. Neurosci. Methods 55:173-82, 1994. 2. Li Y, Kimble J. International C. elegans Meetings, 1995, 1997 3. Hall DH. In C. elegans [H.F. Epstein and D.C. Shakes (eds.)] pp. 395-436, 1995.