Freeze-Cracking of Nematodes: A Method to Expose Interior Worm Tissues for Staining

- Transfer washed C. elegans in liquid onto a glass slide previously coated with poly-lysine, a compound that will help worm cuticles stick to the glass surface. Gently press another glass slide straight down with an edge overhanging the bottom slide. To immobilize the worms without damage, compress the animals avoiding any lateral motions. Then, place the glass slides with sandwiched worms on a metal block inside an insulated laboratory container filled with a freezing agent, such as liquid nitrogen or dry ice.

Metal is an effective temperature conductor ensuring the block surface is cold enough to rapidly freeze the worm's cuticles to both glass surfaces. Once frozen, swiftly separate the glass slides to pull apart or crack open the worm cuticles. Proceed with tissue fixation and staining as disruption of the low permeability cuticle allows chemical fixatives and staining antibodies to access the interior worm tissues. In the following protocol, we will see a detailed demonstration of the freeze-cracking technique and tissue fixation.

- First, prepare a slide to fix the worms. Pipette 30 microliters of poly-L-lysine onto a slide, and then, set a second slide onto the other and ru