Anti cd206 antibody

Hepatic macrophages were isolated from the liver using previously described procedures [9 ,19 (link)]. To identify the M1 or M2 phenotypes, the hepatic macrophages were stained with antibodies against CD80 (1:50, PerCP-eFluor 710-conjugated anti-CD80 antibody; Thermo Fisher Scientific, Rockford, IL, USA) and CD206 (1:50, anti-CD206 antibody; R&D Systems, Minneapolis, MN, USA). The intensity of the fluorescent probe was determined using a Guava easyCyte flow cytometer (Merck Millipore, Burlington, MA, USA) with a 488 nm wavelength laser.

Rat spinal cord (L4–L6) was fixed (4% paraformaldehyde) and embedded (paraffin). After deparaffinization and rehydration processes, sections (5 μm) were obtained, placed (laser confocal small dish), and fixed (ice methanol; 15 min). After blocking, the primary antibodies, anti-Iba1 antibody (1:100, Abcam, Cambridge, MA, USA), anti-CD68 antibody (1: 100, Abcam), anti-CD86 antibody (1: 100, Abcam), anti-CD206 antibody (1:100, R&D Systems, Inc., Minneapolis, MN, USA), were added and incubated (overnight; 4°C). Then, the sections were washed with phosphate-buffered saline (PBS) several times and incubated with the secondary antibody (1 h) counterstained with 4,6-diamidino-2-phenylin-dole (DAPI) (1:1,000; Invitrogen; 5 min; room temperature) before being mounted. PBS was rinsed again, and the images were obtained (a confocal laser scanning microscope; Olympus, Tokyo, Japan). Fluorescent intensity was finally calculated.

After deparaffinization and rehydration through standard protocols, the paraffin sections were washed by PBS. Then sections were alternately bathed in boiling sodium citrate buffer for 20 min. After returning to room temperature, sections were washed. The membrane was permeated in 0.3% PBST (100 ml PBS, 0.3 ml Triton X-100) for 20 min. Sections were washed and then blocked by donkey serum at 37°C for 30 min. Sections were incubated by primary antibodies overnight at 4°C, including anti-F4/80 antibody (1:100, abcam), anti-INOS antibody (1/100, abcam), and anti-CD206 antibody (1:100, R&D). After incubtion the sections were restored to room temperature, washed by PBS, and then incubated by secondary antibodies, including donkey secondary antibodies to rat (1:2,000, Alexa Fluor^®488, abcam), donkey secondary antibodies to rabbit (1:2,000, Alexa Fluor^®647, abcam), and donkey secondary antibodies to goat (1:2,000, Alexa Fluor^®555, abcam). After antibody incubation, sections were washed and finally sealed in a sealant containing DAPI. Images were acquired through a Leica confocal laser scanning microscopy (Leica).

Quercetin, SRT1720 HCI (SIRT1 agonist) and EX527 (SIRT1 antagonist) were purchased from Selleck Chemicals (Houston, TX, USA). Phorbol myristate acetate (PMA), lipopolysaccharide (LPS) and cromolyn sodium were purchased from Sigma Aldrich (St. Louis, MO, USA). Clodronate liposomes were obtained from liposoma B. V (Netherlands). Fetal bovine serum (FBS) was purchased from Gibco (Carlsbad, CA, USA). RPMI 1640 basal culture medium and penicillin/streptomycin were provided by HyClone (Logan, UT, USA). Anti-SIRT1 (Cat# ab189494), anti-TGF-β1 (Cat# ab92486), and anti-tryptase (Cat# ab2378) antibodies were purchased from Abcam Biotechnology (Cambridge, MA, USA). Anti-PDGFRα (Cat# 3174) antibody was purchased from Cell Signaling Technology (CA, USA). Anti-NFkB p65 (acetyl Lys310) antibody (Cat# GTX86963) was purchased from Genetex (CA, USA). Anti-CD206 antibody (Cat# AF2535) was provided by R&D Systems (MN, USA). Anti-β-actin antibody (Cat# 66009-1-Ig) was purchased from Proteintech (Chicago, IL, USA). Anti-IL-1β (Cat# GB11113), anti-TNF-α (Cat# GB13188-2), anti-IL-6 (Cat# GB11117), anti-MCP-1 (Cat# GB11199), anti-F4/80 (Cat# GB11027), anti-F4/80 (GB11119) and anti-IL-10 (Cat# GB11108) antibodies were obtained from Servicebio (Wuhan, China).