Anti iba 1

After the pigs were sacrificed, spinal cord sections (1 cm) from the central DSCI lesion region were collected and embedded in paraffin. Spinal cord sections (5 μm thick) from each specimen were deparaffinized with xylene and incubated in graded concentrations of ethanol. They were then washed with phosphate-buffered saline (PBS) for 3 × 5 min. The sections were incubated for blocking with a blocking solution (0.1% Triton X-100 in PBS and 10% normal goat serum) at room temperature for 2 h. The sections were incubated overnight with primary antibodies at 4°C. The primary antibodies used were anti-GFAP (1: 2000, GeneTex), anti-Iba-1 (1:200, Affinity), and anti-NeuN (1:100, Abcam). After rinsing with PBS for 3 × 5 min, the sections were incubated with secondary antibodies for 2 h at room temperature. The secondary antibody used in this study was goat anti-rabbit antibody (Alexa Fluor^®594) (1:200, Abcam). Following three rinses with PBS, a drop of antifade mounting medium containing DAPI (Solarbio Biotechnology, China) was placed on each slide. Finally, a coverslip was placed on top of each sample. Immunofluorescence imaging was carried out using an Olympus fluorescence microscope.

The rats were euthanized using isoflurane anesthesia after the aforementioned tests. The lumbar segments (L3–L5) of whole spinal cords (n = 6/group) were homogenized and centrifuged at 16,000 g and 4°C for 15 min. Equal amounts of protein (50 μg) in supernatants were denatured, resolved by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE, 5% stacking gel, and 10%–12% separating gel), and then electrotransferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Massachusetts, USA). Nonspecific protein binding on the membranes was blocked with 5% nonfat milk in tri-buffered saline and Tween 20 (TBST) for 2 h at room temperature and then incubated with the primary antibodies: rabbit anti-c-Fos, anti-Iba-1, anti-PKCε, anti-STAT3 (1 : 1000; Affinity Biosciences, OH, USA), anti-IL-6 (1 : 1000; Abcam, MA, USA), anti-GFAP, and anti-GAPDH (1 : 1000; Cell Signaling Technology, MA, USA). The blots were washed with TBST and then probed with secondary HRP goat anti-rabbit IgG (1 : 10,000) in 5% nonfat milk in TBST for 1 h at room temperature. The proteins of interest on the blots were visualized using an ECL reagent (Affinity Biosciences) and photographed using an X-ray film. The protein band intensity was quantified using the ImageJ software (National Institutes of Health, MD, USA).