Bx60 32fb2 a03

Immunofluorescence staining was performed as previously described.20 (link) Briefly, the cells cultured on cover slips were fixed, permeabilized with 0.5% Triton X-100, and incubated with anti-CD163 and anti-CD206 polyclonal antibodies (Proteintech, China. Lot: 00091171 and 00089604, respectively) (1:500) overnight at 4°C. Subsequently, the slides were incubated with secondary antibodies conjugated with Alexa Fluor 488 AffiniPure goat anti-mouse IgG (H+L) (FcMACS, China. Lot: 136908), goat anti-mouse IgG (H+L) CoraLite594 (Proteintech, China. Lot: 20000154), and goat anti-rabbit IgG (H+L) R-PE conjugate (Proteintech, China. Lot: 20000129) (1:1000). Nuclei were stained using 4ʹ,6-diamidino-2-phenylindole (DAPI) for 3 min, and the cells were incubated in the dark for 3 min. The slides were washed with PBS four times, for 5 min each. Then, the slides were sealed with sealing solution containing a fluorescence quencher and observed and imaged under a fluorescence microscope. Immunofluorescence was examined under an epi-fluorescence microscope (Olympus, BX60-32FB2-A03). By changing the filters, the different secondary antibodies could be identified in double-stained sections. Images were captured with a digital camera (Olympus, DP50).