Western blots were performed to measure the relative protein expression of IRF8 (a critical regulatory factor for pro-inflammatory macrophage/microglia activation)15 (link),16 (link) in resected SG at the different time points post-bleomycin instillation. In preparation for Western blot, bilateral SGs were processed using a lysis buffer made from a 100:1 dilution of radioimmunoprecipitation (RIPA) buffer and a protease inhibitor cocktail (Sigma-Aldrich, Atlanta, GA, USA). This mixture was then homogenized, centrifuged and stored at −80°C until use. Protein concentration was measured by the BCA protein assay method using bovine serum albumin (BSA) as standard (Thermo Scientific). The proteins were loaded onto a 10% SDS-PAGE gel along with protein standards (Bio-Rad Laboratories) in a separate lane for electrophoresis and then transferred to polyvinylidene fluoride membrane. The membrane was probed with mouse antibodies against IRF8 (1:500, Santa Cruz Biotechnology) and GAPDH (1:1000, Santa Cruz Biotechnology) and secondary antibody of goat anti-mouse (1:5000, Pierce Chemical). The protein signals were detected by enhanced chemiluminescence reagent (Pierce Chemical) and analysed using UVP BioImaging Systems. The densitometric result of IRF8 was reported as the ratio to GAPDH and then finally normalized to age-matched sham rats.