594 goat anti mouse

The sections from the spinal cord tissues acquired 21 days after SCI were used for immunofluorescence staining as previously described with minor modifications. Briefly, the 10-µm sections were dried for 1 h at room temperature and incubated with blocking solution (5% goat serum in phosphate buffered solution-tween 20) for 2 h at room temperature. Then, the sections were incubated at 4 °C overnight with the following primary antibodies: anti-NeuN antibody (1:300, Abcam, USA) and anti-Nrf2 (1:100, Abcam, USA). The next day, after being washed three times with phosphate buffered solution, the sections were incubated with the following secondary antibodies (488 goat anti-rat IgG, 1:500, Invitrogen, California, USA, and 594 goat anti-mouse, 1:500, Invitrogen, California, USA) for 2 h at room temperature. The sections were then incubated with DAPI for 15 min and sealed with a coverslip. The images were visualized using a fluorescent microscopy (Olympus, Tokyo, Japan). The Image J software was used for analyzing the overlap coefficient of HO-1 with neurons.