Immunoblotting assay

Animals were anaesthetised with 2% isoflurane followed by cervical dislocation. Dorsal root ganglia (DRG), spinal cord (SC) and thalamus were immediately excised to extract proteins on day 14 after the first acid saline injection. Total proteins were prepared by homogenising tissue in lysis buffer containing 50 mM Tris-HCl pH 7.4, 250 mM NaCl, 1% NP-40, 5 mM EDTA, 50 mM NaF, 1 mM Na₃VO₄, 0.02% NaN₃ and 1×protease inhibitor cocktail (AMRESCO). The extracted proteins (30 µg per sample assessed by BCA protein assay) were subjected to 8% SDS-Tris glycine gel electrophoresis and transferred to a PVDF membrane. The membrane was blocked with 5% non-fat milk in TBS-T buffer (10 mM Tris pH 7.5, 100 mM NaCl, 0.1% Tween 20), incubated with primary antibody (Alomone Labs Ltd) in TBS-T with 1% bovine serum albumin, and incubated for 1 hour at room temperature. Peroxidase-conjugated anti-rabbit antibody (1:5000) was used as a secondary antibody. The bands were visualised by an enhanced chemiluminescent substrate kit (Pierce) with LAS-3000 Fujifilm (Fuji Photo Film Co Ltd). Where applicable, the image intensities of specific bands were quantified with National Institutes of Health (NIH) ImageJ software (Bethesda, MD, USA).