Retro-Orbital DiI Labeling for Ocular Motor Nuclei

For DiI fills, embryos or larvae were fixed at relevant developmental stages in 4% PFA at 2 hr at RT, or overnight at 4°C. Specimens were then carefully placed in a small petri dish with a sylgard-coated bottom containing PBS and secured with the eye facing up using a Tungsten minuten pin. The eye was carefully removed from the orbit with sharp forceps and DiI (D3911, Invitrogen) was applied to the tissue at the corner of the orbit, where the oculomotor and trochlear nerves enter, using a blunt microinjection needle held by a micromanipulator. DiI was left to bind to the tissue for 3–5 min, after which embryos were stored overnight in PBS at 4°C. The following day, brains were dissected from whole larvae and mounted for imaging. For live retro-orbital dye fills, ocular motor nuclei were retrogradely labelled as previously described (Greaney et al., 2017 (link)). Crystallised fluorescently-conjugated Alexa Fluor 647 dye (10,000 molecular weight, Thermo Fisher D-22914) was applied unilaterally to the orbit of anesthetised fish at four dpf and the side of incision and dye application was recorded for each larva. Larvae were returned to the incubator at 28.5°C to recover overnight before imaging at 5 dpf.