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Advances in C. elegans Specimen Preparation for TEM Using Microwave and Fast Freeze Techniques

Published online by Cambridge University Press:  02 July 2020

D.H. Hall
Affiliation:
Center for C. elegans Anatomy, Albert Einstein College of Medicin, Bronx, NY10461
T. Starich
Affiliation:
Biological Sciences Center, University of Minnesota, Minneapolis, MN55108
J. Shaw
Affiliation:
Biological Sciences Center, University of Minnesota, Minneapolis, MN55108
V. Gobel
Affiliation:
Massachusetts General Hospital, Boston, MA02114
J. Fleming
Affiliation:
Massachusetts General Hospital, Boston, MA02114
C. Norris
Affiliation:
Department of Biology, Johns Hopkins University, Baltimore, MD02118
C-c. Huang
Affiliation:
Department of Pathology, Robert Wood Johnson Medical School, Piscataway, NJ08854
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Abstract

The nematode C. elegans is a simple model for genetic studies of cell and tissue development. There is a need to improve the preservation of embryonic and early larval stages, during which nematode tissues elaborate and separate. However, these stages are particularly resistant to fixation and embedment due an impenetrable eggshell and larval cuticle. Their small size at these ages precludes mechanical cutting, which has been used successfully for immersion fixation of older stages. Here we compare the quality of preservation under three rather different regimes: using laserholes to permeabilize the eggshell during the primary fixation step, using microwave energy to enhance the first fixation step, or using fast freezing and freeze substitution to circumvent the standard immersion procedure. Vancoppenolle et al (2000) have recently demonstrated very good results through enzymatic weakening of the eggshell prior to immersion fixation. Their data are comparable to what we achieve by either the laserhole or microwave methods.

Type
Recent Techniques for the Fixation and Staining of Biological Samples (Organized by M. Sanders and K. McDonald)
Copyright
Copyright © Microscopy Society of America 2001

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References

References:

Vancoppenolle, B et al. (2000) Evaluation of fixation methods for ultrastructural study of C. elegans embryos. Microscopy Research & Technique 49: 212216.3.0.CO;2-1>CrossRefGoogle Scholar