Immunohistomount

Longitudinal tissue sections (20 μm) were incubated with primary and secondary antibodies (Abs) shown in Table 2.
For double and triple immunofluorescence labeling, sections were blocked with 5% normal goat serum for 1 hour at room temperature (RT) and then incubated overnight at 4°C with a mixture of primary Abs raised in distinct species. Prior to visualization, sections were incubated with fluorophore-conjugated secondary Abs for 2 h at RT. 4',6-Diamidino-2-phenylindole (DAPI) (10 μg/mL in PBS, Sigma) was used for visualizing nuclei. Coverslips were mounted on slides using mounting medium (ImmunoHistoMount, Santa Cruz). Sections were examined using an LSM 780 Confocal Microscope (Carl Zeiss, Germany). Only HNu-cells that displayed clearly outlined nuclei were evaluated. The mean intensity of labeling (semi-quantitative analysis of GAP-43) 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 longitudinal histological sections collected at 50-μm increments extending from the contusion center (observed area, 2 mm²) of the SCI or Th8 vertebral level for sham group.

Transverse tissue sections were incubated with primary and secondary antibodies (Abs) shown in Table 2. For immunofluorescence labeling, the sections were blocked with 5% normal goat serum for 1 h at room temperature (RT) and then incubated overnight at 4°C with a primary Abs. Prior to visualization, the sections were incubated with corresponding fluorophore-conjugated secondary Abs for 2 h at RT. 4′,6-Diamidino-2-phenylindole (DAPI) (10 μg/mL in PBS, Sigma) to visualize nuclei. Coverslips were mounted on slides using mounting medium (ImmunoHistoMount, Santa Cruz). The sections were examined using a LSM 780 Confocal Microscope (Carl Zeiss, Germany). The mean intensity of labeling (semi-quantitative analysis of GFAP and Iba1) 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 of the SCI. The following areas were selected for a semiquantitative immunohistochemical evaluation of glial cells: the ventral horn (VH), the main corticospinal tract (CST), ventral funiculi (VF), the area around the central canal (CC) and the dorsal root entry zone (DREZ).

NKA-ATPase localization was determined using immunolabeling according to the protocol adapted from Ituarte et al. (2016) (link). Before to block non-specific binding, the sections were placed in a citrate-buffered solution, pH 6, heated up in the microwave (10 min) to expose the epitopes. Afterwards, they were incubated in a humidity chamber at 4 °C overnight with the rabbit anti-Na⁺/K⁺-ATPase H300 primary antibody (Santa Cruz Bio-technology) diluted in phosphate buffered saline containing Régilait^® (PBS-R 0.5%) at 4 µg/mL. To remove the excess of antibody, the sections were washed in PBS before being incubated with the secondary antibody (Alexa Fluor^®488 donkey anti-rabbit, Invitrogen) at 10 µg/mL in PBS-R 0.5% for 1 h at room temperature. Sections were mounted in an anti-bleaching medium (ImmunoHistoMount, Aqueous-based Media, Santa Cruz Bio-Technology). Negative control was made with the secondary antibody alone and positive control was made by Western Blot with specific NKA antigen (Ituarte et al., 2016 (link)).

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).