Anti mouse igg conjugated with alexa 546

For immunofluorescence labeling, the sections were blocked with 5% normal goat serum for 1 h at room temperature (RT) and then incubated separately overnight at 4°C with a primary antibody against ionized calcium binding adaptor molecule 1 (Iba1) (Abcam, 1:300) and glial fibrillary acidic protein (GFAP) (Millipore, 1:200). Prior to visualization, the sections were incubated with a fluorophore-conjugated secondary antibody (anti- mouse IgG conjugated with Alexa 546, Invitrogen, 1:200) for 2 h at RT. 4′,6-Diamidino-2-phenylindole (DAPI) (10 μg/mL in PBS, Sigma) was used to visualize nuclei. Coverslips were mounted on slides using a mounting medium (ImmunoHistoMount, Santa Cruz). The sections were examined under a LSM 780 Confocal Microscope (Carl Zeiss, Germany). The total intensity of labeling (semi-quantitative analysis of Iba1 and GFAP) was analyzed using Zen 2012 Software (Carl Zeiss). All sections were imaged in the z-plane using identical confocal settings (laser intensity, gain, and offset). Measurements were obtained from transverse histological sections collected at 5-mm increments extending from the contusion center (observed area, 2 mm²) of the SCI. The following areas were selected for semiquantitative immunohistochemical evaluation of glial cells: the main corticospinal tract (CST), ventral funiculi (VF), and the ventral horn (VH).