Following antibodies were used in this study: choline acetyltransferase (ChAT) to detect cholinergic motor neurons, Nrf2 and HO-1 to investigate Nrf2/HO-1 signaling pathway, IL-1β to examine proinflammation, and glial fibrillary acidic protein (GFAP) to detect astrocytes in the lumbar spinal cord.
Immunohistochemical staining was conducted according to a published method [28 (link)] with modifications. Briefly, the spinal cord sections, which are described above, were immersed in 0.3% hydrogen peroxide (H2O2) for 20 min and then in 5% normal donkey serum for 30 min. After washing, the sections were incubated in each diluted primary antibody overnight at 4 °C as follows: rabbit anti-ChAT (1:200; Chemicon, Temecula, CA, USA), rabbit anti-Nrf2 (1:500; Proteintech, Rosemont, IL, USA), rabbit anti-HO-1 (1:500; Abcam, Cambridge, UK), rabbit anti-IL-1β (1:200; Abcam), and mouse anti-GFAP (1:800; Abcam). After washing, the sections were transferred to biotinylated horse anti-mouse or goat anti-rabbit IgG (diluted 1:200; Vector, Burlingame, CA, USA) for two hours, and followed by a streptavidin peroxidase complex (diluted 1:200; Vector) for one hour. Thereafter, the sections were washed and visualized with 0.2% 3, 3′-diaminobenzidine tetrahydrochloride. Finally, the sections were covered with DPX (Sigma Aldrich, St. Louis, MO, USA).
Immunohistochemical staining was conducted according to a published method [28 (link)] with modifications. Briefly, the spinal cord sections, which are described above, were immersed in 0.3% hydrogen peroxide (H2O2) for 20 min and then in 5% normal donkey serum for 30 min. After washing, the sections were incubated in each diluted primary antibody overnight at 4 °C as follows: rabbit anti-ChAT (1:200; Chemicon, Temecula, CA, USA), rabbit anti-Nrf2 (1:500; Proteintech, Rosemont, IL, USA), rabbit anti-HO-1 (1:500; Abcam, Cambridge, UK), rabbit anti-IL-1β (1:200; Abcam), and mouse anti-GFAP (1:800; Abcam). After washing, the sections were transferred to biotinylated horse anti-mouse or goat anti-rabbit IgG (diluted 1:200; Vector, Burlingame, CA, USA) for two hours, and followed by a streptavidin peroxidase complex (diluted 1:200; Vector) for one hour. Thereafter, the sections were washed and visualized with 0.2% 3, 3′-diaminobenzidine tetrahydrochloride. Finally, the sections were covered with DPX (Sigma Aldrich, St. Louis, MO, USA).