Goat anti cd206

Double Immunofluorescence staining was performed on fixed frozen brain sections as previously described.^{17 (link)} The 8-μm thick slides were rinsed with PBS, and permeabilized with 0.3% Triton X-100 for 15min at room temperature. Next, slides were incubated with blocking solution (95% PBS, 5% normal donkey serum, and 0.05% Triton X-100) for 2 hours. Then, the slides were incubated using the following: goat anti-NeuN (neuronal nuclei; 1:300), mouse anti-Iba1 (ionized calcium-binding adaptor molecule 1; 1:300), rabbit anti-FKN (1:100), rabbit anti-CX3CR1 (1:100), rabbit anti-CD68 (cluster of differentiation 68; 1:150), rabbit or goat anti-CD206 (the mannose receptor; 1:200), goat anti-CD206 (1:200), rabbit anti-CD163 (1:100) or rabbit antihemoglobin (1:100; Abcam) at 4 °C overnight. After 3 consecutive washes in PBS (10 minutes each time), the sections were incubated with appropriate fluorescent-conjugated secondary antibodies (1:200; Jackson ImmunoResearch) for 2 hours at room temperature. The number of CD68⁺Iba1⁺ or CD206⁺Iba1⁺ positive cells in 3 different fields of the right basal cortex was identified and counted from 5 random coronal sections per rat, and positive cells were quantified under microscope fields at ×400 magnification.

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.