Mannose receptor cd206

Tissues were embedded in Optimal Cutting Temperature Compound (OCT, Fisher Scientific) and then sectioned into 10 μm slices. Sections were stained with OX-42 (1:1000, Abcam), mannose receptor (CD206) (1:500, Abcam), GFAP (1:1000, Abcam), and NeuN (1:1000, Abcam) at 4°C overnight. Appropriate fluorescence conjugated secondary antibodies (Jackson Immunoresearch, West Grove, PA) were incubated for 1 h at room temperature. Leica DMi8 (Leica Microsystems, Germany) was used to image the peri-ventricular region. M2 microglia activated cells were counted on 6 sections in every group over a 20 × microscopic field and measured as cells/field, as described ^{31 (link)}.

Lung sections were deparaffinized and antigen retrieval was performed by heating sections to 90°C for 20 min in Target Retrieval Solution (DAKO, Carpinteria, CA) using a water bath. Endogenous peroxidases were quenched using Peroxidazed-1 (Biocare Medical, Concord, CA). To block non-specific binding, sections were next incubated with Rodent Block M (Biocare Medical) for 20 min at room temperature. Sections were then incubated with antibodies to either Mannose Receptor (CD206) (1:2500, Abcam, Cambridge, UK) overnight at 4°C, Arginase-1 (Arg-1) (1:300, Proteintech Group, Chicago, IL) for 45 min at room temperature, or with monocyte chemotactic protein-1 (MCP-1) (1:1000, Abcam) overnight at 4°C, all diluted in Renaissance Diluent (Biocare Medical). Sections were then rinsed and Rabbit on Rodent HRP polymer (Biocare Medical) was next applied for 25 min at room temperature and binding was visualized using diaminobenzidine (DAB) (Biocare Medical).

Protein concentrations for immunoblot (Lekic et al., 2011 (link)) were determined by DC protein assay (Bio-Rad, Hercules, CA). 30 µg protein per sample were loaded into wells of 4–20% gels, ran for 30 minutes at 50V then 90 minutes at 125V, then transferred onto nitrocellulose membranes at 0.3A for 120 minutes (Bio-Rad). Membranes were incubated for 2 hours in 5% non-fat milk in Tris-buffered saline containing 0.1% Tween20. Then the following primary antibodies were incubated overnight: anti-PPARy (1:1000; Santa Cruz, Dallas, Texas), CD36 (1:500, Santa Cruz, Dallas, Texas), and mannose receptor (CD206) (1:500, ABcam, Cambridge, Massachusetts). Secondary antibodies (1:2000; Santa Cruz Biotechnology, Santa Cruz, CA) were then applied to the membranes and incubated for 2 hours and then processed with the ECL plus Kit (GE Healthcare and Life Science, Piscataway, NJ). β-actin was used as an internal control against the anti-body (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA). ImageJ software (4.0, Media Cybernetics, Silver Spring, MD) was used to analyze the relative density of the resultant protein immunoblot images as described (Tang et al., 2004 (link)).