Mouse anti il 6

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).

Slide-mounted tissue sections were washed with PBS for 5 min, incubated in
3% hydrogen peroxide for 20 min and washed 3 times in PBS. They were then heated
in 1% antigen-unmasking solution (Vector Laboratories) for 20 min at 90°C,
incubated for 1 h in permeabilization buffer (10% goat serum, 0.1% Triton
X-100 in PBS) and incubated overnight at 4°C with rabbit anti- IL-1β
(1:100) or mouse anti - IL-6 (1:100) or mouse anti-TNFα (1:50) primary antibody
(Abcam) in antibody solution (5% goat serum, 0.05% Triton X-100 in PBS).
Next day, the sections were washed with PBS and incubated 1 h at room temperature with
biotinylated goat anti-rabbit, (1:400), or biolinylated goat anti- mouse (1:400)
antibodies (Vector Laboratories) in antibody solution. Sections were then washed in PBS,
incubated in avidin-biotin complex mixture (ABC,1:100) for 1 h at room temperature, washed
again and developed with diaminobenzidine solution (DAB), washed with PBS, dried and cover
slipped with vectamount mounting medium. For immunostaining of RFP sections were
pre-treated in the same way and incubated with rabbit anti-DsRed (1:1000) primary antibody
(Abcam) over night at 4°C, washed with PBS and incubated with biotinylated goat
anti-rabbit antibody, 1:400, (Vector Laboratories) for 1 h at room temperature. Sections
were washed with PBS, dried and cover slipped with Vectashield aqueous mounting medium
with DAPI.