Dmi4000b fluorescence microscope system

To enable us to mimic Schwann cell precursor migration from ganglia, Schwann cell precursor migration was measured using reaggregated precursors essentially as previously described [22] (link). Reaggregates were formed by plating Schwann precursor cells on Corning Ultra Low Attachment dishes (Corning, NY, USA) for 4 h and on Petri dishes for 20 h with gentle agitation every 4–6 h. Individual Schwann cells were allowed to migrate out of the reaggregates in the presence or absence of 20 ng/ml of PDGFalpha (Peprotech, Rocky Hill, NJ, USA), NRG1 (Bio-Techne, Minneapolis, MN, USA), NT3 (Peprotech), or IGF1 (Life Technologies) on type I collagen-coated dishes. After incubation at 37 °C for 6 h, cells were fixed with 4% PFA solution (Nacalai Tesque), permeabilized with 0.1% Triton X-100 solution (Nacalai Tesque), and blocked with Blocking One reagent (Nacalai Tesque). Blocked samples were immunostained with an anti-p75^NTR antibody and then with a fluorescence-labeled secondary antibody, and were mounted in Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA). The fluorescence images were captured with a DMI4000B fluorescence microscope system (Leica, Wetzlar, Germany) and analyzed with AF6000 software (Leica). 0.4% Trypan blue-stained attached cells routinely accounted for less than 5% of all cells after incubation for 6 h with each growth factor.

Cells on coverslips or dishes were normally fixed with 4% paraformaldehyde (PFA). Four percent PFA followed by 100% cold methanol was used for myelin segment staining (Chan et al., 2004 (link); Yamauchi et al., 2012 (link); Miyamoto et al., 2013 (link), 2018). Cells were permeabilized with phosphate-buffered saline (PBS) containing 0.1% Triton X-100, blocked with Blocking One reagent, and incubated first with primary antibodies and then with fluorescence-­labeled secondary antibodies. The coverslips or dishes were mounted with Vectashield reagent with 4’,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA). The fluorescence images were captured with a DMI4000B fluorescence microscope system (Leica, Wetzlar, Germany) and analyzed with AF6000 software (Leica). Culture dishes (Rnd2 shRNA and control shRNA) were harvested on different days, and at least three sets were prepared for experiments. All photos in the figures are representative of multiple experiments.