Fitc f4 80 monoclonal antibody

Prior to co-culture, apoptosis of A549 cell-acquired azide groups was induced by incubation with staurosporine at 5 µM for 24 h 34 (link)-36 (link). Peritoneal macrophages were seeded onto 35-mm glass-bottom dishes or six-well plates at a density of 1×10⁶ cells in 2 mL of media and incubated for 24 h. Macrophages were then incubated with apoptotic cells for up to 2 h at 37 °C. After incubation, macrophages were washed twice with DPBS (pH 7.4) and stained with FITC-F4/80 monoclonal antibody (Abcam, Cambridge, UK). A549 cells, fluorescently labeled according to the manufacturer's protocol using DiD dye (Invitrogen) prior to the induction of apoptosis, were used as controls. Cellular imaging and flow cytometry analysis were performed by the same methods as described above.

The retrieved liver tissues were fixed in 4% (volume/volume) buffered paraformaldehyde solution and embedded in OCT tissue compound (Sakura, Tokyo, Japan). They were then frozen in a dry ice bath, cut on a cryostat (6 μm in thickness), picked up on slides with poly-D-lysine, and dried at 45 °C under light protection. Fluorescence images were obtained using a FluoView FV10i confocal scanning microscope (Olympus) equipped with a water-immersion objective lens (UplanSApo 60×/NA=1.2) and a diode laser. The excitation wavelength was 405 nm (17.1 mW) for the DAPI stain and 635 nm (9.5 mW) for the DBCO-Cy5. One day after injection of DBCO-Cy5, the organs of DBCO-Cy5-injected mice, including the liver, lung, spleen, and kidneys were also harvested and examined histologically to determine cytotoxicity of DBCO-Cy5 to the distant organs. The sliced tissues (6 μm) were stained with hematoxylin and eosin and observed with a light microscope (BX51; Olympus). Images were photographed on a digital camera photomicroscope (DP71; Olympus). To examine distorted signals, the sliced liver tissues (6 μm) were stained with FITC-F4/80 monoclonal antibody (Abcam, Cambridge, UK). Fluorescence images were observed using IX81-ZDC focus drift compensating microscope (Olympus, Tokyo, Japan).