C. elegans Gonad Dissection and Freeze Crack for Immunofluorescence and DAPI Staining

J Vis Exp. 2022 Sep 16;(187). doi: 10.3791/64204.

Abstract

The C. elegans germline makes an excellent model for studying meiosis, in part due to the ease of conducting cytological analyses on dissected animals. Whole mount preparations preserve the structure of meiotic nuclei, and importantly, each gonad arm contains all stages of meiosis, organized in a temporal-spatial progression that makes it easy to identify nuclei at different stages. Adult hermaphrodites have two gonad arms, each organized as a closed tube with proliferating germline stem cells at the distal closed end and cellularized oocytes at the proximal open end, which join in the center at the uterus. Dissection releases one or both gonad arms from the body cavity, allowing the entirety of meiosis to be visualized. Here, a common protocol for immunofluorescence against a protein of interest is presented, followed by DAPI staining to mark all chromosomes. Young adults are immobilized in levamisole and quickly dissected using two syringe needles. After germline extrusion, the sample is fixed before undergoing a freeze crack in liquid nitrogen, which helps permeabilize the cuticle and other tissues. The sample can then be dehydrated in ethanol, rehydrated, and incubated with primary and secondary antibodies. DAPI is added to the sample in the mounting medium, which allows reliable visualization of DNA and makes it easy to find animals to image under a fluorescent microscope. This technique is readily adopted by those familiar with handling C. elegans after a few hours spent practicing the dissection method itself. This protocol has been taught to high-schoolers and undergraduates working in a research lab and incorporated into a course-based undergraduate research experience at a liberal arts college.

Publication types

  • Video-Audio Media
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Caenorhabditis elegans Proteins* / metabolism
  • Caenorhabditis elegans* / metabolism
  • Ethanol / metabolism
  • Female
  • Fluorescent Antibody Technique
  • Germ Cells
  • Gonads / metabolism
  • Indoles
  • Levamisole / metabolism
  • Meiosis
  • Nitrogen / metabolism
  • Staining and Labeling

Substances

  • Caenorhabditis elegans Proteins
  • Indoles
  • Levamisole
  • Ethanol
  • DAPI
  • Nitrogen