Ox 42

Immunohistochemistry (IHC) of inflammatory cells was performed on three animals per group. Extracted brains were cut into 3-mm thick coronal slices and immersed in a 20% sucrose solution for 2 hrs at 4°C. Brain slices were embedded in OCT (Sakura Finetek Inc., Torrance, CA, USA) and stored at −80°C. For immunostaining, 10 μm coronal sections were obtained by using a cryostat (Tissue-Tek Cryo3; Sakura Finetek Inc.). Sections were further incubated with 3% H₂O₂ and 10% methanol in PBS, to block endogenous peroxidase. Sections were subsequently incubated overnight at 4°C with primary antibodies against CD68 (ED-1, 1:50 dilution; Abcam) and CD11b (OX42, 1:50 dilution; Abcam), followed by incubation with a biotin-conjugated secondary anti-rabbit antibody (1:200; Vector Laboratories Inc., Burlingame, CA, USA) for 1 hr and streptavidin-conjugated peroxidase (Vecstatin Abc kit, Vector Laboratories Inc.) for 30 min. The slices were developed by incubation (3 min.) with DAB 0.05% (w/v; Dako, Glostrup, Denmark) and visualized under an IX-51 microscope (Olympus Life and Material Science Europe GMBH, Hamburg, Germany), which was attached to a DS-U2 LCD camera (Nikon Instruments Inc., Melville, NY, USA). All slides were evaluated by a blinded, trained observer. Cells were counted in five representative fields of 192 μm² in the infarcted area.

The OVA antigen was purchased from Sigma (A8040, USA) and aluminum hydroxide as adjuvant was from Kelong chemical factory (lot number 201110328, Chengdu, Sichuan, China). Enzyme-linked immunosorbent assay (ELISA) kits, specific for IgE (sIgE), interleukin 4 (IL-4), interferon-γ (IFN-γ), TNF-α, and IL-6 were purchased from Abcam (England). Antibodies against SP, GFAP, and CD11b (OX42) and rabbit anti-mouse secondary antibody were also purchased from Abcam, England. Where mentioned, biotin-conjugated rat anti-mouse antibodies were purchased from BD Pharmingen, Beijing, China. Sodium citrate buffer (0.01 M, pH 6.0) was prepared for dilution, where needed. The microscopic image acquisition and analysis system were, respectively, from BA200 Digital and Image-Pro Plus 6.0 (Media Cybernetics, USA).

Under anesthesia, rats were perfused with 4% paraformaldehyde through the ascending aorta. The L6-S1 spinal cord section was removed and postfixed in paraformaldehyde for 30 min. The spinal cord was then transferred to 30% sucrose for dehydration at 4°C. Tissues were sectioned (25 μm thickness) and processed for immunofluorescence staining. Sections were blocked for 1 h and then incubated with primary antibodies against Iba-1 (1 : 200; Abcam), CD11b (OX-42, 1 : 400; Abcam), TNF-α (1 : 200; Bioworld), and IL-1β (1 : 500; Abcam), overnight at 4°C. After that, the sections were incubated in Cy3 or Alexa-488 conjugated secondary antibodies (Jackson Laboratories, Bar Harbor, ME, USA) for 1 h at room temperature. A Leica fluorescence microscope (Leica DFC350 FX camera; Wetzlar, Germany) was used to measure and image the stained section. To quantify Iba-1, OX-42, TNF- α, and IL-1β expression in the L6-S1 SDH, the fluorescence intensity of each area was analyzed with ImageJ software. To verify the specificity of primary antibodies, immunostaining was also performed in parallel but without primary antibodies (data not shown).

Primary rat microglia were prepared as previously described, with sight modifications [15 (link)]. In short, the whole brain tissues of postnatal days one to two from SD rats were triturated. The dissociated cells were passed through a 100 μm pore mesh, pelleted by centrifugation at 1500 rpm for 5 min, and resuspended in culture medium. Cells were seeded on poly-D-lysine-precoated cell culture flasks in high-glucose Dulbecco's Modified Eagle's Medium (DMEM, Gibco, USA) containing 10% fetal calf serum, 100 U/ml penicillin, and 100 mg/ml streptomycin (Gibco, USA). Cultures were maintained at 37°C in a humidified atmosphere of 5% CO₂/95% air. After the glial cells formed a confluent monolayer (10-14 days), the microglial cells were separated from the astrocytes by shaking for 5 h at 110 rpm. The microglial cells were seeded into 6-well culture plates at a density of 10⁵ cells/cm². After 24 h of culture, the cells were starved overnight and then subjected to treatments. The primary microglia were pretreated with LiCl (1 mM) for 30 min and then incubated with LPS (10 ng/ml) for 24 h. The purity of the microglia was confirmed to be >98% using immunofluorescence staining for OX-42 (Abcam, Hong Kong, China) and was calculated as follows: number of OX − 42 − positive cells/number of DAPI − positive cells.

The rat spinal cord L4-6 was dehydrated through a sucrose gradient. The tissues were surrounded by OCT, and then dry ice and absolute ethanol were poured into the Petri dish to quick-freeze the tissue. After quick freezing, place the tissue on the microtome and cut out 15 μm tissue sections. The experimental area was circled with an immunohistochemical pen, fixed with PFA for 30 min, and blocked with goat serum for 30 min. Shake off the goat serum, drop the primary antibody p-p38 (Abcam, USA) and OX42 (Abcam, USA), and incubate at 4°C for one night. The next day, rinse with PBST. Add fluorescent secondary antibody, incubate at 37°C for 30 min in the dark, and then rinse with PBST. After counterstained by DAPI, wash with PBST 3 times. The slides were mounted with antifluorescence quenching mounting medium and photographed by using a fluorescence microscope.