Twee-Foton Laser Axotomie: Een methode om axonen in zebravisembryo's te verwonden en axonale herstel te observeren

- Place embryos of interest into phenyl thiourea, PTU, Ringer's solution to inhibit pigment formation, which begins at 24 hours post fertilization. This makes the developing anatomy more visible, which is necessary for this procedure. Transfer a tricaine anaesthetized embryo into an imaging chamber. Place a glass slide on top and view it under your microscope. Focus on the axon to be injured, which is visible under a 488 nanometer laser because it transgenetically expresses GFP.

Take a before image of the site. Next, view the axon with a 910 nanometre laser which causes the GFP to fluoresce red. Increase the intensity of this laser to injure the target area without affecting the surrounding tissue. This process is called axotomy. Take a new image with the first laser to confirm the axotomy, which appears as scattered debris. In the following protocol, we will perform an axotomy of peripheral sensory axons in zebrafish embryos using a two-photon laser.

- If a custom built two-photon scope is not available in your laboratory, the Zeiss 510 confocal two-photon microscope can also be used to sever axons. We begin by placing the mounted embryo onto the stage and bringing it into focus u