Pe conjugated anti f4 80 antibody

All plasmids in this study were constructed and sequenced to confirm their identity. The Tim-4, Tim-4^AAA, Tim-4^TL, Tim-4-GPI, and Anxa5-GPI plasmid used in this study have been previously described^{33 (link)}. Tim-4^ΔIgV lacking the IgV domain of Tim-4 (residues 25–134) and Tim-4^ΔMucin without the mucin domain (residues 135–270) were constructed into the pDisplay vector using a PCR-based method. The antibodies used in the study were anti-HA antibody (Santa Cruz, SC-7392), PE-conjugated anti-F4/80 antibody (Biolegend, 123110), Alexa Fluor 405-conjugated anti-mouse antibody (Invitrogen, A31553), Alexa Fluor 488-conjugated anti-mouse antibody (Invitrogen, A-11029), and Alexa Fluor 555-conjugated anti-mouse antibody (Invitrogen, A-21422). Polystyrene beads (FluoSpheres carboxylate beads, Invitrogen, F8826), FITC-labeled E. coli (E-2861), S. aureus (S-2851), and zymosan A (Z-2841) bioparticles were purchased from Invitrogen. Alexa Fluor 488-conjugated LPS (L-23351) was purchased from Molecular Probes. LPS from E. coli O55:B5 (L2880) was purchased from Sigma Aldrich. About 6.0–8.0-μm streptavidin-coated polystyrene particles (SVP-60-5) were purchased from Spherotech. For IgG opsonization, FITC-conjugated streptavidin antibody (200-402-095) was purchased from Rockland Immunochemicals. Dexamethasone (265005) and TAMRA-SE (C1171) were purchased from Merck and ThermoFisher, respectively.

Flow cytometry was carried out to analyze the abundance of the M1 marker CCR7, M2 marker CD206, and general macrophage marker F4/80. After 24 h of culture, BMDMs in the four groups were scraped, washed, blocked for 15 min with Blocking Buffer (Beyotime), and then stained for 1 h with an allophycocyanin (APC)-conjugated anti-CCR7 antibody (1:100, BioLegend, USA) or FITC-conjugated anti-CD206 antibody (1:100, BioLegend, USA). A PE-conjugated anti-F4/80 antibody (1:100, BioLegend, USA) was used to label all macrophages. The cells were analyzed using a BD flow cytometer with FlowJo software.

To evaluate apoptosis of cells in liver tissues, the TUNEL assay was performed. In brief, frozen liver tissue sections (8 μm thickness) were stained with a DeadEnd™ Fluorometric TUNEL System (Promega) according to the manufacturer’s protocols. To identify the types of TUNEL-positive apoptotic cells, immunofluorescence staining was sequentially done using markers for various cells. Alexa 594-conjugated anti-αSMA antibody (1:100, Cell Signaling Technology, cat. No. 36110S), PE-conjugated anti-F4/80 antibody (1:50, BioLegend, cat. No. 123110), APC-conjugated anti-CD26 antibody (1:100, BioLegend; cat. No. 137807) and rabbit anti-mouse cytokeratin 7 antibody (1:100, Abcam; cat. No. ab181598) with Alexa Fluor 647-conjugated goat anti-rabbit IgG antibody (1:200, Abcam; cat. No. ab150083, Lot No. GR3370563-1) were used to mark aHSC, macrophages, and hepatocytes, respectively. After overnight incubation at 4 °C, DAPI staining and mounting were performed on the slides, and fluorescence images of slide were obtained by VECTRA (Perkin-Elmer). Then, images were analyzed by InForm 2.2.1 image analysis software (Perkin-Elmer) followed by quantification of co-localization of TUNEL signals with each cell marker.