Talin clone 8d4

Animals were first sacrificed with an overdose of sodium pentobarbital, and perfused transcardially with PBS followed by 4% PFA, before dissecting out the spinal cord and sciatic nerve. The tissue was cryoprotected in 30% sucrose solution at 4 °C for 3 days, embedded in OCT (RALamb UK) and cut longitudinally using a cryostat into 14 μm sections. Sections were washed in PBS (15 min, room temperature), permeabilized with 0.1% Triton X-100 (15 min, room temperature), blocked with 10% goat serum (1 h, room temperature) and incubated in primary antibodies (diluted in blocking solution) at 4 °C overnight. The following day, sections were washed with PBS, incubated with secondary antibodies (1 h, room temperature) and then mounted in Fluorosave. Primary antibodies were used against: talin (clone 8D4, Abcam, 1:20) and beta III tubulin (Sigma, 1:400).

Cell cultures on coverslips were fixed with 4% paraformaldehyde (PFA), permeabilized with 0.1% Triton X-100, blocked with goat or donkey serum and incubated with primary antibodies at 4 °C overnight. They were then washed and incubated with secondary antibodies for 1 h at room temperature before being mounted on slides. Primary antibodies were used against beta III tubulin (Sigma, 1:400), pY397 FAK (BD Biosource, 1:100), talin (clone 8D4, Abcam, 1:20), GFP (Abcam, 1:500), and mCherry (Clontech, 1:200).