Fitc conjugated cd206

Animal and human tissue samples were fixed with 10% formalin solution (pH 7.4) overnight, embedded on a slide with paraffin and sectioned using standard protocols. Sections were then deparaffinised, incubated in warm 1% citrate buffer for 10 min, cooled at room temperature for 1 hour and then washed with distilled water for 2 min before antigen retrieval. Nonspecific binding sites were blocked by incubation in 1% BSA/PBS for surface molecule staining; to detect intracellular components, blocking was performed with 1% BSA/PBS with 0.1% Triton X-100. Subsequently, rabbit anti-human IL-33 (1:200, Santa Cruz) and FITC-conjugated CD206 (1:50, BD Bioscience) antibodies were added to the sections and incubated at 4 °C overnight. IL-33 signal was detected with goat anti-rabbit IgG Alexa Fluor 555 antibodies (1:500, Invitrogen). Nuclei were counterstained with DAPI (blue). Images were obtained using a fluorescence light microscope (Olympus BX41, Olympus Optical, Tokyo, Japan).

Cell surface and intracellular cytokine staining of monocytes was performed as previously described (46 (link)). Briefly, cultured monocytes or purified monocytes were washed in PBS and stained by PE-conjugated anti-CD80 (BD Pharmingen, 557227), FITC-conjugated anti-CD86 (BD Pharmingen, 555657), PE-conjugated anti-CD163 (BD Pharmingen, 556018), FITC-conjugated CD206 (BD Pharmingen, 551135), PE-Cy7–conjugated HLA-DR (BD Pharmingen, 560651), or APC-conjugated anti-CD42b (BD Pharmingen, 551061) before analysis by FACSCanto. Fixation and permeabilization were performed using Cytofix/Cytoperm Plus kit with GolgiPlug (BD Biosciences) and stained by PE-conjugated anti–IL-1β (R&D Systems, IC201P) and PE-CF594–conjugated anti–IL-10 (BD Horizon, 562400) before analysis. All data acquired from flow cytometry were analyzed with FlowJo (Treestar software).

Phorbol 12-myristate 13-acetate (PMA), Lipopolysaccharides (LPS) and Hoechst 33258 dye were from Sigma-Aldrich (St. Louis, MO, USA). The cytokines for interferon γ (IFNγ), interleukin-4 (IL-4) and interleukin-13 (IL-13) were from R&D Systems (Minneapolis, MN, USA). The antibody for CD163 was from Abcam (Cambridge, MA, USA). The primary antibodies for E-cadherin, GAPDH were from Cell Signaling Technology (Danvers, MA, USA). Antibodies for fluorescein isothiocyanate (FITC)-conjugated CD68, FITC-conjugated CD206, FITC-conjugated CD163, FITC-conjugated HLA-DR and isotype control were from BD Biosciences (San Jose, CA, USA). FITC-conjugated E-cadherin antibody was from BioLegend (San Diego, CA, USA). Hepatocyte maintenance medium (HMM) and SingleQuots were from Lonza (Anaheim, CA, USA).