Rabbit anti interleukin 1β il 1β

Immunohistochemistry was performed according to a previously published protocol, with modification (Jian et al., 2016). Mouse brain tissue was cut into 5-µm-thick paraffin sections and incubated with primary antibodies (rabbit anti-progranulin and goat anti-tumor necrosis factor-α (TNF-α), 1:100, Santa Cruz Biotechnology, Dallas, TX, USA; rabbit anti-inducible nitric oxide synthase (iNOS) and rabbit anti-interleukin-1β (IL-1β), 1:150, Abcam, Cambridge, MA, USA; rabbit anti-matrix metalloproteinase-3 (MMP-3), 1:100, Abcam; rabbit anti-phospho-NF-κB inhibitor α (p-IκBα), 1:100, Abcam) at 4°C for 12 hours. After incubating with secondary antibodies (goat anti-rabbit, 1:200, Abcam; rabbit anti-goat, 1:150, Jackson ImmunoResearch, PA, USA) at 37°C for 1 hour, the sections were stained with a diaminobenzidine kit (ZSGB-Bio, Beijing, China), and observed with an IX-81 microscope (Olympus, Beijing, China) and quantified using Image-Pro Plus 6.0 (Media Cybernetics, Rockville, MD, USA).

Proteins were extracted from the retinas of C57BL/6 mice at specific time points and adjusted for protein content (40 µg). Protein samples were then mixed with 5× SDS sample buffer (Beyotime), separated by SDS polyacrylamide gel electrophoresis, and transferred onto polyvinylidene fluoride (PVDF) membranes (EMD Millipore, Bedford, MA, USA). After being blocked in 5% nonfat dried milk for 2 hours at room temperature, the membranes were incubated overnight at 4°C with primary antibodies. Primary antibodies used in this study included rabbit anti-IκBα, rabbit anti-p-IκBα, rabbit anti-p65, rabbit anti-p-p65, rabbit anti-iNOS, mouse anti-β-actin (Cell Signaling Technology), rabbit anti-interleukin-1β (IL-1β), rabbit anti-Toll-like receptor (TLR)2 (Abcam), goat anti-CD206, rabbit anti-transforming growth factor-β (TGF-β), rabbit anti-TLR4 (Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-Arg-1 (GeneTex), goat anti IL-1RA, goat anti-monocyte chemoattractant protein1 (MCP-1; R&D Systems, Minneapolis, MN, USA). Membranes were washed and incubated for 2 hours with secondary antibodies conjugated with horseradish peroxidase (Cell Signaling Technology). We used enhanced chemiluminescence (ECL) western blotting detection solutions (EMD Millipore) to display the immunoreactive bands.