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.