Apc conjugated anti f4 80 antibody

To quantify the populations of Th1, Th2, Th17 and Foxp3-positive Treg cells, murine splenocytes and stromal vascular cells were stimulated with 25ng/mL phorbol myristate acetate (PMA) and 250ng/mL ionomycin (both from Sigma-Aldrich, St. Louis, MO, USA) and Golgi Stop (BD Biosciences, San Diego, CA, USA) in a 24-well plate and incubated for 4 hours. The stimulated splenocytes were stained with PerCP-conjugated anti-CD4 antibody (eBiosciences), then fixed and permeabilized using the Cytofix/Cytoperm Plus Kit (BD Biosciences) following the manufacturer’s protocol. Splenocytes were then reacted with fluorescein isothiocyanate (FITC)-conjugated anti-IL-17 antibody (eBiosciences). For analysis of the number of Treg cells, splenocytes were labeled with anti-CD4 and anti-CD25 antibodies, followed by fixation, permeabilization, and intracellular staining with anti-Foxp3 antibody as per the manufacturer’s instruction. To quantify the population of F4/80+CD11c+CD206- (M1) and F4/80+CD11c-CD206+ (M2) cells, mouse splenocytes were stained using allophycocyanin (APC)-conjugated anti-F4/80 antibody, FITC-conjugated anti-CD11c antibody and Phycoerythrin (PE)-conjugated anti-CD206 antibody (eBiosciences). All samples were analyzed by a FACS Calibur device (BD Pharmingen) [48 (link)].

The sections of liver tissue were blocked with 2% BSA for 30 minutes at RT, and then incubated with APC-conjugated anti-F4/80 antibody (eBioscience, San Diego, CA) at RT for 30 minutes. After washing in PBS for three times, Each sections was observed under a confocal laser scanning microscope (Olympus IX71).

Syk inhibitors SykI and BAY 61-3606, PI3K inhibitor LY294002, ERK inhibitor U0126, JNK inhibitor SP600125, and p38 inhibitor SB203580 were purchased from Calbiochem-Merck (Darmstadt, Germany). Raf-1 inhibitor GW5074, methyl-β-cyclodextrin (MβCD), and water-soluble cholesterol were obtained from Sigma-Aldrich.
Antibodies against phospho (p)-Zap-70 (Tyr319)/Syk (Tyr352), p-Akt (Tyr308), p-c-Raf (Ser338), p-ERK1/2 (Thr202/Tyr204), p-JNK (Thr183/Tyr185), JNK, p-p38 (Thr180/Tyr182), p-IKKα/β (Ser176/180), p-NF-κBp65 (Ser536), p-IκBα (Ser32), IκBα, p-c-Jun (Ser63), c-Jun, p-c-Fos (Ser32), and c-Fos were purchased from Cell Signaling (Beverly, MA, USA). Anti-Syk, anti-β-actin, HRP-conjugated anti-rabbit IgG, and HRP-conjugated anti-mouse IgG antibodies were purchased from GeneTex Inc. (Irvine, CA, USA). Blocking antibodies against CR3 (clone 5C6) and Dectin-1 (clone 2A11) were purchased from Serotec (Oxford, UK). Antibodies for cell surface staining, anti-CD11b (clone M1/70), anti-CD18 (clone GAME-46), and APC-conjugated anti-F4/80 antibody were obtained from eBioscience (San Diego, CA, USA), and anti-Dectin-1 (clone 218820) was purchased from R&D Systems (Minneapolis, MN, USA).