Imagequant las 4000 mini system

CNS, microglial cells and neurons protein extract analysis were performed from 5 and 10 nerve cords respectively T24 h post-injury with RIPA buffer. For each experimental condition, SDS-PAGE was conducted (4–12% polyacrylamide gel) using 30 µg of protein extract homogenized (v/v) in 2X Laemmli sample buffer and loaded in the gel wells, further details are found in Supplementary Method S6. After migration of proteins the gel is transferred in a membrane and was incubated for 1 hour at RT in blocking buffer (0.05% Tween 20 w/v, 5% milk powder w/v in 0.1 M PBS, pH 7.4) and then overnight at 4 °C in rabbit polyclonal anti-TGF-β1 (1/200, Abcam, Cambridge, UK) in blocking buffer. After rinsing three times with PBS-0.05% Tween 20 for 15 minutes, the membrane was incubated for 1 hour at RT in secondary goat anti-rabbit polyclonal antibody conjugated with horseradish peroxidase (1:20,000, Jackson Immunoresearch, Cambridgeshire, UK) in PBS-0.05% Tween 20. Finally, after another rinsing with PBS, immunoreactive bands were revealed using the ECL Kit SuperSignal West Dura ChemoLuminescent Substrate (Thermo Fisher Scientific, Waltham MA, USA). Chemiluminescence analyses were performed by ImageQuant LAS-4000 mini system (Fujifilm, Tokyo, Japan).

Immunoblot analyses were carried out as described previously.^{36 (link)} Briefly, cells were lysed with RIPA buffer containing 1 mM phenylmethylsulfonyl fluoride (Sigma), 1 mM sodium orthovanadate (Na₃VO₄) and a complete protease inhibitor cocktail (Roche) for 10 min on ice. The membrane was treated with primary antibodies (Abs) at 4 °C overnight, followed by incubation with secondary antibodies for 2 h. The primary antibodies were purchased as follows: HoxD10 (E-20) was from Santa Cruz Biotechnology (Santa Cruz, CA), phospho-ERK1/2 was from Cell Signaling Technology (Beverly, MA), and α-tubulin was from Sigma Aldrich. The signals were developed using ECL reagents (GE Healthcare, Little Chalfont, UK) and were visualized using an ImageQuant LAS4000 mini system (Fujifilm, Tokyo, Japan).