Gap 43

Mounted tissue sections were equilibrated to room temperature, washed in PBS for 2 × 5 minutes, permeabilised in 0.1% Triton x-100 in PBS for 20 minutes and washed for 2 × 5 minutes in PBS. Sections were blocked in blocking buffer (75 μl; 0.5% bovine serum albumin (g/ml), 0.3% Tween-20, 15% normal goat/donkey serum (Vector Laboratories) in PBS) in a humidified chamber for 30 minutes and incubated with primary antibody (RNA-binding protein with multiple splicing (RBPMS), 1:500, ThermoFisher, #ABN-1376; growth associated protein-43 (GAP-43), 1:400, ThermoFisher, #33–5000; AAV, 1:200, ThermoFisher, #AB-PA1–4106) diluted in ADB (15% normal goat serum in place of bovine serum albumin) overnight at 4°C. The following day, slides were washed for 3 × 5 minutes in PBS and incubated with secondary antibody (Mouse IgG 488, 1:400, ThermoFisher, #A11001; Guinea Pig IgG 546, 1:400, ThermoFisher, #A-11074) diluted in ADB for 1 hour at room temperature. Slides were washed for 3 × 5 minutes in PBS, mounted in Vectorshield mounting medium containing DAPI (Vector Laboratories) and stored at 4°C before microscopic analysis. Negative controls including omission of primary antibody were included in each run and were used to set the background threshold levels prior to image capture.