Volocity

All microscopy samples were embedded in homemade mowiol (Sigma-Aldrich) mounting medium, and images were acquired on an SP8 laser-scanning microscope (Leica Microsystems) equipped with STED depletion lasers. All images besides the STED superresolution images were acquired at room temperature with a 63× oil immersion objective (high-contrast Plan Apochromat 63× 1.40 NA Oil CS2) using the standard software (LAS X; Leica Microsystems) that came with the microscope. The LIF files were then opened with Volocity (PerkinElmer), exported as Bitmap files from Volocity, and transferred into Illustrator (Adobe) via cut and paste from Photoshop (Adobe). Where necessary for visibility in the final illustrator file, brightness was increased uniformly across the entire image and across all conditions shown using Photoshop.

In order to avoid potential regional differences in maturation, the medial portion of the MNTB was selected for analysis (Fig. 3B; Rodríguez-Contreras et al., 2008 (link); Ford et al., 2009 (link)). A 40×/NA1.4 oil lens attached to an LSM510 confocal microscope (Carl Zeiss) was used to acquire z-stacks of 8-bit, 512 × 512 pixel images. Image stacks (x, y, z voxel of 0.58, 0.58, 1.13 mm) were imported into Volocity (Perkin Elmer, Waltham, MA) for analysis using available image-segmentation and colocalization routines (Rodríguez-Contreras et al., 2005 (link), 2008 (link)). Cell density estimates were corrected to exclude cells touching the borders of the image in x, y, and z planes, and to take into account differences in z-stack volume. Cell density values were reported in cells/mm³. Brightness and contrast modifications were performed in Volocity or in Adobe Photoshop (San Jose, CA) during the preparation of figures.