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

Overview

This video describes freeze-cracking, a method to make C. elegans tissues accessible for antibody staining by disrupting the cuticle.

Protocol

This protocol is an excerpt from Zhang et al, The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analysis and Imaging, J. Vis. Exp. (2017).

Antibody staining of the C. elegans intestine

1. Fixation
   1. Take a clean glass slide and use poly-L-lysine to generate a thin film for worms to stick on. Place 30 µL 0.1-0.2% poly-L-lysine on the slide and place a second slide on the poly-L-lysine drop to make a "sandwich". Then rub the slides gently a few times to wet the entire surfaces of both and let them air dry for 30 min. Label the frosted side of the slides with pencil.
      NOTE: 0.2% poly-L-lysine aliquots of 200 µL were made by dissolving the powder in dH₂O; this can be stored at -20 °C. Use high molecular weight poly-L-lysine for improved sticking of the worms. The concentration of poly-L-lysine is also critical. Too low concentrations will not allow worms to stick but too high concentrations may generate fluorescence background signal. A too thick film may loosen up in its entirety.
   2. Place a flat metal block firmly on the bottom of a container (e.g. a polystyrene container) filled with liquid nitrogen.
      NOTE: One can use dry ice instead, but liquid nitrogen keeps a metal block more stable on the container bottom and chills well.
   3. Collect worms either by washing them off their plates with M9 or pick different stage worms (either eggs, L1, L2, L3, or L4 larval stage worms) onto each slide. Typically, pick ~100 larvae and embryos or ~20 adults to each slide. Place 10 µL washed off worms onto the middle of the slide or pick eggs/worms into 10 µL M9 or 10 µL 1x PBS (phosphate buffered saline).
      1. Use a pipette to spread out large numbers of worms to avoid clumping.
         Note: Mixed populations of washed off worms, due to crowding and different thickness of stages, do not stick well and are less effectively freeze-cracked (see below), therefore picking stage-specific worms (or synchronized populations) gives superior results. Larvae stick better than adults and more animals can be placed per slide.
      2. Before picking worms onto slides, transfer them to a Nematode Growth Medium (NGM) plate without OP50 bacteria. Excess bacteria adherent to the worms can also interfere with sticking. Take care that worms do not dry out.
   4. Gently place (drop) a 22 mm × 22 mm coverslip cross-ways on top of the collected worms such that its edges hang over on at least one side of the slide. Press straight down gently but firmly with one or two fingers on the coverslip. Avoid shearing that will damage tissue integrity.
   5. Immediately and gently transfer the slide to the metal block in liquid nitrogen and let it sit for about 5 min to freeze. Then "flick off" the coverslip in one swift move by using the overhanging edge.
      NOTE: This step must be done decisively and while the slide is frozen to achieve "cracking" of the cuticle. Caution: Please follow the PPE (Personal Protection Equipment) guidelines when working with liquid nitrogen.
   6. Immerse the freeze-cracked slides into a methanol-filled glass Coplin jar for 5 min at -20 °C. Then transfer to an acetone-filled glass Coplin jar for another 5 min at -20 °C.
      NOTE: Methanol and acetone should be stored in -20 °C for at least 30 min before use. After fixation, slides can be stored at -20 °C.
      CAUTION: methanol and acetone are toxic.
   7. Remove slides from jar and let them air dry at room temperature (RT) before use.

Materials

Name	Company	Catalog Number	Comments
Antibody staining
poly-L-lysine	Sigma	P5899
Methanol	Fisher Scientific	A452-4
Acetone	Fisher Scientific	A949SK-4
Permount	Fisher Scientific	SP15-100
Microscope slides	Fisher Scientific	4448
Microscope coverslips (22x22-1)	Fisher Scientific	12-542-B
M9 Medium			lab made
OP50 bacteria	CGC