Epidermal Laser Wounding: Inducing Localized Damage to Examine Repair Processes in C. elegans
Transcrição
– To begin, transfer young adult worms onto a fresh agarose pad on a slide. Then add a few drops of 12 millimolar levamisole solution para paralyze the worms. Cover the worms with a coverslip and wait for a few minutes. Place the slide under a spinning disk confocal microscope and focus on the lateral outermost surface of the worm.
The epidermis of an adult worm is a thin single-layered structure consisting of multi-nucleated syncytia that result from cell fusion events. The apical surface of epidermis secretes a flexible collagenous cuticle, while the basal surface is covered by a thin layer of extracellular matrix called the basal lamina.
Next, wound the apical surface of the epidermis with a laser. Cytoplasm from the damaged cells may leak out, appearing as a bubble at the wound site.
Laser irradiation has an advantage over other techniques, such as needle wounding, as it causes localized disruption of the cuticle and epidermal cells, without damaging internal tissues. In the example protocol, we will see the process of laser wounding de C. elegans.
– To precisely wound the nematodes, this protocol uses a femtosecond laser attached para spinning disk confocal microscope. Line or point scans using femtosecond lasers, such as those de two-photon microscopes, should also suffice.
– Begin by collecting 10 young adults on an agar pad. Paralyze them de a two microliter drop of 12 millimolar levamisole solution. Then cover them with a cover slip and wait a few minutes while they paralyze.
– It is essential that an animal be completely paralyzed. 12 millimolar levamisole will not affect the wound response. However, other methods of immobilization can also be used.
– Ensure that the femtosecond laser power is set para 140 milliwatts, as measured before the objective, and the laser repetition rate is at 80 megahertz. Now, locate the worms under a spinning disk confocal microscope. Using a 100x objective with a high numerical aperture, focus on the lateral anterior or posterior syncytial epidermis of the target worm.
Focusing on the apical surface of the epidermal cell, shine the laser for para two 200 millisecond pulses separated by 20 milliseconds. This should be sufficient para wound the epidermis. Wait a moment and observe the local disruption of cytoplasm, which will appear as bubbling. If a fluorescent marker is present, localized bleaching can be observed.
