A Workflow for High-pressure Freezing and Freeze Substitution of the Caenorhabditis elegans Embryo for Ultrastructural Analysis by Conventional and Volume Electron Microscopy.

The free-living nematode Caenorhabditis elegans is a popular model system for studying developmental biology. Here we describe a detailed protocol to high-pressure freeze the C. elegans embryo (either ex vivo after dissection, or within the intact worm) followed by quick freeze substitution. Processed samples are suitable for ultrastructural analysis by conventional electron microscopy (EM) or newer volume EM (vEM) approaches such as Focused Ion Beam Scanning Electron Microscopy (FIB-SEM). The ultrastructure of cellular features such as the nuclear envelope, chromosomes, endoplasmic reticulum and mitochondria are well preserved after these experimental procedures and yield accurate 3D models for visualization and analysis ( Chang et al., 2020 ). This protocol was used in the 3D reconstruction of membranes and chromosomes after pronuclear meeting in the C. elegans zygote ( Rahman et al., 2020 ).