Airyscan super resolution microscope

Spinal cord tissue for these analyses was collected at 30 WPI and sectioned horizontally at 30μm thickness using CryoJane tape transfer method. Immunostaining for ⅙ sampling interval sections was performed to detect PRV-GFP labeled fibers. For quantification, five to six random optical fields within the lesion site per section were imaged using Zeiss LSM 900 with Airyscan super resolution microscope. Antibody source and the dilutions were used as listed in Supplemental Table 1. For each optical field, 24 μm Z stack images (0.4 μm Z-step) were captured using 60X oil objective. PRV-GFP labeled filament volumes were manually traced using Imaris v9.6 filament manual tracing software (Oxford Instruments, Abingdon, United Kingdom). Brain sections were sectioned coronally at 30μm using a sliding microtome. Immunostaining (all sections containing motor cortex, no sampling) was performed to detect PRV-GFP cell bodies. All PRV⁺ cell bodies within the motor cortex were counted manually. Quantification was performed at 20X magnification using ZEISS Axio Imager II light microscope with an Apotome2 image processor. Image acquisition and quantifications were performed by investigators blinded to the experimental groups.

Spinal cord tissue for these analyses was collected at 30 WPI and sectioned horizontally at 30-μm thickness using the CryoJane tape transfer method. Immunostaining for 1/6 sampling interval sections was performed to detect PRV-GFP labeled fibers. For quantification, five to six random optical fields within the lesion site per section were imaged using Zeiss LSM 900 with an Airyscan super-resolution microscope. The antibody source and the dilutions were used as listed in Supplemental Table 1. For each optical field, 24 μm Z stack images (0.4 μm Z-step) were captured using a 60X oil objective. PRV-GFP labeled filament volumes were manually traced using Imaris v9.6 filament manual tracing software (Oxford Instruments, A Abingdon, United Kingdom). Brain sections were sectioned coronally at 30 μm using a sliding microtome. Immunostaining (all sections containing motor cortex, no sampling) was performed to detect PRV-GFP cell bodies. All PRV⁺ cell bodies within the motor cortex were counted manually. Quantification was performed at 20X magnification using a ZEISS Axio Imager II light microscope with an Apotome2 image processor. Image acquisition and quantifications were performed by investigators blinded to the experimental groups.