Lsm710

2 month old mice were anesthetized with isoflurane and the sciatic nerve exposed by a small incision on the skin. The nerve was crushed with a pair of fine (#55) forceps for 20 s and the crush site marked using activated carbon powder (Bauder and Ferguson, 2012 (link)). 3 days later the mice were euthanized by CO₂ and sciatic nerves obtained for analysis. Sciatic nerves were fixed in 4% paraformaldehyde for 3 hr, then washed with PBS, immersed in 30% sucrose in PBS, cryopreserved in OCT compound (TissueTek) and cryosectioned at 10 μm thickness. Samples were immunostained with anti-SCG10 (1:3000) (Novus Biologicals STMN2 NBP1-49461). After staining, multiple images along the nerve were taken using a 10X objective (Zeiss LSM710) and montaged using Photoshop (Adobe). Representative images are shown in Figure 6D. We used Metamorph software to measure SCG10 staining fluorescence intensity in tdTomato positive axons (Cre-expressing neurons). At each distance point, at least 10–20 regions with tdTomato signal were randomly selected in the red channel and regions of interest (ROI) defined. These regions were then transferred to the green channel and the average intensity of green fluorescence measured. Average intensity from all the regions at each distance point was then normalized to the average intensity immediately proximal to the crush site.

Embryos were fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.76 mM KH₂PO₄, pH 7.4) overnight at 4 °C. The numbers of cells in the neuromasts and PLLP were counted by DAPI staining as described previously^{18 (link),48 (link),49 (link)}. EdU incorporation was performed as described previously^{47 (link)}. Embryos were dechorionated and 30.5-hpf embryos were immersed in 500 μM EdU solution on ice for 30 min. After washing, embryos were incubated at 28.5 °C for 1 h and fixed at 32 hpf in 4% PFA overnight at 4 °C. The EdU signals were detected using a Click-iT EdU Alexa Fluor488 Imaging Kit (Invitrogen), according to the manufacturer’s instructions. Embryos were embedded in 0.5–1% low melting point agarose (Invitrogen). Confocal images were captured with a Zeiss LSM700 or Zeiss LSM710 microscope and processed using Adobe Photoshop Elements 12 and Fiji^{57 (link)}.