Immunohistochemistry

Nerve sections were washed in immunostaining buffer (PBS together with 0.2% Triton‐X (Sigma‐Aldrich), 0.002% sodium azide (Sigma‐Aldrich) and 0.25% Bovine Serum Albumin) before the addition of horse serum (1:20 in immunostaining buffer) for 45 min. The blocking serum was then removed and sections were incubated in anti‐neurofilament antisera (1:1000 in immunostaining buffer) (Neurogentec) overnight at 4°C. The sections were then washed with immunostaining buffer before addition of the secondary antibody, Dylight anti‐mouse IgG 549 (1:200) (Vector Laboratories) and incubation at room temperature for 45 min. Sections underwent a final wash with immunostaining buffer before mounting with Vectashield (Vector Laboratories).