Freeze-Cracking of Nematodes: A Method to Expose Interior Worm Tissues for Staining
Transcript
– 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 rub them together to spread the solution evenly onto both. Now, peel apart the two slides and let them air dry for 30 minutes.
Next, place a flat metal block into a container filled with liquid nitrogen. Wash the plates with M9 solution and transfer about 10 microliters of M9 containing the worms. Alternatively, pick the worms and add M9 to the slide.
Once the worms are transferred, gently drop a coverslip crossways onto them such that the coverslip overhangs one edge. Then, gently press the coverslip without making any lateral movements.
Now, immediately transfer the slide to the metal block in liquid nitrogen and let it freeze. After five minutes, flick off the coverslip using the overhanging edge. Do this swiftly to get the proper crack of the cuticle.
Next, immerse the slide in a methanol-filled glass Coplin jar at minus 20 degrees Celsius. After five minutes, transfer the slide to an acetone-filled glass Coplin jar for another five minutes at the same temperature. The slides can be stained directly or stored at minus 20 degrees Celsius.
