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

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.