Confocal lsm710 airyscan

VGlut1 and VGAT synaptic boutons were imaged with a Confocal LSM710/Airyscan (Zeiss). Z-stack images were taken with an average thickness of 18 μm with a step size of 0.2 μm. Multi-fluorescent orthogonal 3D image analysis and visualization were performed using Zen 3.2 software. The average number of vGlut1 or VGAT boutons opposed to motor neurons from L1-4 spinal sections was calculated by analyzing at least 10 motoneurons per sample.

DRG cultures, DRG sections, and sciatic nerves sections were imaged using a Nikon Ti2 Eclipse epifluorescent microscope with 4×, 10×, and 20× objectives. Spinal cord sections and hind paw sections were imaged using a Dragonfly spinning disk microscope (Andor Technologies) with a 20× or 63× objective. Sections were stitched using the Fusion software with 10% overlap between tiles. Synaptic contacts to motoneuron were imaged with a Confocal LSM710/Airyscan (Zeiss) with a 63× objective. Multi-fluorescent orthogonal 3D image analysis and visualization were performed using Zen 3.2 software.