Fv1000 bx61

For perfusion-fixation, mice were deeply anesthetized with ketamine and transcardially perfused sequentially with cold saline (0.9% NaCl), then 4% paraformaldehyde in saline. Tissues were dissected, postfixed for 1 h at 4°C (except overnight for brain and spinal cord), washed in PBS (3.8 mm NaH₂PO₄, 16.2 mm Na₂HPO₄, and 15 mm NaCl/1 l), cryoprotected overnight at 4°C in 30% sucrose in PBS, and embedded in OCT. Fixed cochleas, were decalcified for one week (Bas et al., 2019 (link)) before embedding in OCT.
Cryosections were cut on a Leica CM1900, sensory ganglia and lingual tissue to 20-μm thickness, spinal cord and brain to 40 μm. Sections, mounted on slides, were permeabilized (0.1% Triton X-100 in PBS), blocked (10% normal donkey serum) and incubated in diluted primary antibodies overnight at 4°C. After washing for 1 h in PBS, secondary antibody was incubated for 1–2 h. Sections were mounted under Fluoromount G (SouthernBiotech). Antibodies used and their concentrations are in Table 1.
Imaging was on an Olympus Fv1000 BX61 upright or an Olympus Fv1000 IX81 inverted laser scanning confocal microscope. Multichannel images were captured and were adjusted, only for brightness, in Photoshop. No contrast enhancement was applied. All images are shown at, or smaller than captured size.

Confocal microscopy was performed on an FV1000-BX61 laser-scanning confocal microscope using an UPlanSApo 60×/NA 1.35 objective (all Olympus). Movies were captured using the FV1000-IX81 microscope using a PlanApo N 60×/NA 1.42 objective (all Olympus). Macrochaetes were imaged for all experiments. z-stack image generation and brightness and contrast adjustment were performed using ImageJ (NIH) without any nonlinear adjustments. Gaussian filter was applied to generate still images from time-lapse imaging of S2 cells.
The ‘tip index’ was determined as follows (see also supplementary material Fig. S1). A line scan was performed from the base of the bristle to the distal tip to obtain a plot profile using ImageJ. Subsequent analyses were performed using Excel (Microsoft). The maximum intensity (100% intensity) and bristle length (Position[Max]) were determined from the line scan, and pixels that exceeded 50% intensity were identified. The tip index was defined as the relative position of the pixels that exceeded 50% intensity along the proximal-distal axis of the bristle; the full bristle length was defined as 100. Statistical analyses (Student's t-test) were performed using Excel.

After isolation, O/PE were fixed for 1 h at room temperature (RT) in PBS containing 4% paraformaldehyde (PFA) (Merck, Darmstadt, Germany). Embryos were then stored at 4 °C in 1% PFA for a maximum of one week. O/PE were then transferred through three drops of PBS/BSA (0.1% BSA, referred to as the PBS/BSA next in the text) and permeabilization was performed. Cells were stored in PBS containing 0.5% Triton X-100 (Sigma-Aldrich, Saint-Louis, MO, USA) for 1 h at RT and were then washed in PBS/BSA two times for 5 min. Following overnight incubation at 4 °C in rabbit polyclonal anti-GPx4 antibody conjugated with FITC (#orb8194, 1:50, Biorbyt Ltd., Cambridge, UK), O/PE were washed six times for 5 min in PBS/BSA at RT. DNA was stained using Hoechst 33,342 (10 μL mL⁻¹ in PBS; Sigma-Aldrich, Saint-Louis, MO, USA) for 5 min at RT. Embryos were again washed in PBS/BSA and mounted on the slides using Vectashield (Vector Laboratories, Burlingname, CA, USA). Finally, we examined and photographed O/PE under a confocal microscope (FV-1000 BX61; Olympus, Tokyo, Japan), as was described previously by Baran et al., 2013 [75 (link)].

Larvae to be imaged were transferred to N-phenylthiourea to inhibit pigmentation at 24 h post-fertilization (hpf). Prior to imaging, larvae were anesthetized with MS222 and embedded in 1% low-melting-point agarose in embryo medium containing MS222. All images of the spinal cord were taken laterally, such that anterior was to the left, and dorsal was at the top. To investigate OPC migration at 3 and 4 dpf in vivo, Tg (olig2: EGFP) transgenic zebrafishes were imaged under a 10 × objective lens and a 40 × objective lens (FV1000 BX61; Olympus, Tokyo, Japan) at 2 μm intervals, locating within a frame long and wide with the cloacal pores at the central point of the spinal cord. After imaging, fish were removed from the agarose and placed in fresh embryo medium for further growth. Photomontages were assembled with Adobe Photoshop CS5 (Adobe Systems, San Jose, CA, United States).