Rabbit anti ntn 1

After rats were perfused with ice-cold PBS (0.1M, pH 7.4) at 24 h post-operation, the ipsilateral cortex were collected and stored in −80 °C freezer until use. Western blot was performed as described previously (Tong et al., 2017 ). After protein samples preparation, equal amounts of protein (50 µg) were separated by SDS-PAGE gel electrophoresis, and then transferred onto nitrocellulose membranes. Membranes were blocked and incubated with the following primary antibodies overnight at 4 °C: rabbit anti-NTN-1 (1:800, Abcam, USA), rabbit anti-UNC5B (1:1000, Abcam, USA), rabbit anti-PPARγ (1:500, Abcam, USA), rabbit anti-NFκB P65 (1:1000, Abcam, USA), mouse anti-IL-6 (1:1000, Abcam, USA), goat anti-TNF-α (1:1000, Abcam, USA), rabbit anti-ICAM-1 (1:1000, Santa Cruz Biotechnology, USA), and rabbit anti-myeloperoxidase (MPO, 1:1000, Santa Cruz Biotechnology, USA). β-actin was used as the internal loading control. Then, membranes were incubated with horseradish-peroxidase conjugated secondary antibodies (Santa Cruz Biotechnology, USA) for 1 h at room temperature, The immunoblots were probed with an ECL Plus chemiluminescence reagent kit (Amersham Biosciences, USA). The relative density of protein was analyzed by ImageJ software (ImageJ 1.5, NIH, USA).

Rats were anesthetized with isoflurane at 24 h post SAH, and perfused with cold 120 mL of PBS (0.1M, pH 7.4). After removal of brain, the ipsilateral/left cerebral cortex tissues that facing blood clots were instantly collected and stored in −80 °C freezer until use. Western blot was performed as described previously (Tong et al., 2016 ). After protein samples preparation, equal amounts of protein (50 μg) were separated by SDS-PAGE gel electrophoresis, and then transferred onto nitrocellulose membranes. Membranes were blocked and incubated with the following primary antibodies overnight at 4 °C: rabbit anti-NTN-1 (1:800, Abcam), rabbit anti-APPL-1 (1:2000, Cell Signaling), rabbit anti-phospho-AKT (Ser473) (1:1000, Cell Signaling), rabbit anti-AKT (1:1000, Cell Signaling), rabbit anti-Bcl-2 (1:500, Santa Cruz Biotechnology), and rabbit anti-CC-3 (1:1000, Abcam). β-actin was used as an internal loading control. Then, membranes were processed with horseradish-peroxidase conjugated secondary antibodies (Santa Cruz Biotechnology) for 1 h at room temperature. Immunoblots were probed with an ECL Plus chemiluminescence reagent kit (Amersham Biosciences). The relative density of protein was analyzed by ImageJ software.

Double fluorescence staining was performed as described previously (Huang et al., 2015 (link)). Frozen coronal sections (10 μm) were blocked with 5% donkey serum for 1 h and incubated at 4°C overnight with primary antibodies: rabbit anti-NTN-1 (1:500, Abcam), and mouse anti-NeuN (1:500, Abcam) followed by incubation with appropriate fluorescence-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA) for 2 h at room temperature. Negative control staining was conducted by omitting the primary antibody. Finally, slides were covered with DAPI (Vector Laboratories, Inc.). The sections were visualized under a fluorescence microscope Leica DMi8. Microphotographs were analyzed with LASX software.