Fluoromount mounting medium with dapi

To confirm successful intraspinal delivery of LV‐ChABC, sections spanning the injury epicenter were stained for C‐4‐S using tyramide signal amplification, to reveal stub epitopes which are only present after degradation of chondroitin sulfate glycosaminoglycans (CS‐GAGs). Sections were rehydrated with PBS and incubated at room temperature in the following: hydrogen peroxide (0.3%, 20 min), mouse monoclonal anti‐C‐4‐S (MP Biomedicals; 1:5000, overnight), biotinylated secondary antibody (anti‐mouse biotin; Vector Laboratories, USA; 1:400, 2 h), ABC reagent (ThermoFisher Scientific, USA; 1:250, 30 min), biotinyl tyramide (PerkinElmer Life Sciences, Boston, MA; 1:75, 10 min), and extra‐avidin FITC (Sigma‐Aldrich; 1:500, 3 h). Slides were coverslipped using Fluoromount mounting medium with DAPI (#CO‐4959‐52, Invitrogen). Representative images of C‐4‐S immunostaining for each treatment group were obtained using a confocal microscope (Zeiss, LSM710), using identical exposure and settings.

Every other section of the spinal cord (20 μm apart) was immunostained for CTB. The slides were washed with PBS, blocked for 1 h in 3% BSA in PBS (Bovine Serum Albumin; #A3059; Sigma‐Aldrich) and incubated overnight in the primary antibody goat anti‐CTB (#7032A10; List Biological Laboratories, Inc.) 1:4000 in 0.2% Triton‐x100 and 1% BSA in PBS. Slides were then washed in PBS and incubated with the secondary antibody Alexa Fluor 488 (#A11055; Invitrogen) 1:1000 in 0.2% Triton‐x100 and 1% BSA in PBS for 3.5 h protected from light. After PBS washes, the slides were coverslipped using Fluoromount mounting medium with DAPI (#CO‐4959‐52, Invitrogen). All sections with positive motor neurons were imaged using a VS120 Olympus slide scanner using DAPI and FITC filters to visualize cell nuclei and CTB, respectively. The software QuPath was used to draw individual spinal levels and count neurons in each spinal level. The presence of the fiducial marks determined the limits of C4 and C8 levels, and the other levels were determined by distance. For each animal, the total number of CTB positive neurons present in each spinal level (from C2 to T2) was calculated.