780 laser confocal microscope

FITC-KB or FITC-LPS was incubated with LPS or KB for 30 min and added to TLR4^−/− HCs (2×10⁵/ml) for 1 h. Cells were fixed in 4% paraformaldehyde, permeabilized by 0.01% Triton and incubated with primary antibodies for ASGPR1, EEA1, Rab7 and LAMP1, followed by staining with Alexa Fluor 647 or CY3-labled anti IgG (H+L). Nuclei were stained with DAPI and fluorescence images were captured with a ZEISS 780 laser confocal microscope (Germany).

iPSCs were seeded on 12-well chamber slides (Ibidi, 81201) and cultured for 2 days. Cells were then rinsed with Dulbecco’s phosphate-buffered saline (DPBS) and fixed with ice-cold 4% paraformaldehyde (PFA) for 15 min. Cells were then incubated in 5% normal serum and 1% Triton X-100 in PBS for 45 min at room temperature. Primary antibodies were diluted in 5% normal serum (Merck) and 1% Triton X-100, and incubation was carried out overnight at 4 °C. Primary antibodies were detected by incubating with appropriate fluorescent secondary antibodies for 90 min at room temperature. For nuclear staining, 10 mg/ml Hoechst 33342 (Thermo Fisher Scientific) was added for 10 min after the incubation with secondary antibodies. For labeling TRA-1-60 membrane protein, cells were first incubated in 5% normal serum and 0.025% Triton X-100 for 45 min, then stained with mouse anti-TRA-1-60 IgM diluted in 5% normal serum and 0.025% Triton X-100 overnight at 4 °C. The secondary antibody was added for 90 min at room temperature. For double labeling with other primary antibodies, cells were fixed again with ice-cold PFA for 10 min, and staining was performed as described above. Images were obtained using a Zeiss 780 laser confocal microscope (Zeiss).

Frozen sections of liver tissue were prepared immediately after sampling. The sections were fixed with ice-cold acetone for 15 min, sealed with 10% goat serum at 4 °C for 30 min, and permeabilized with 0.2% Triton X-100 for 15 min. This was followed by incubation of the sections with antibodies against cytokeratin 18 (1:100, Abcam, USA) or F4/80 (1:250, CST, USA) at 4 °C overnight. The sections were incubated with Cy3-conjugated secondary antibody (Beyotime, 1:400, China) and dyeing with TUNEL and DAPI (Beyotime, China). Changes in cellular calcium ion concentration were analyzed via immunofluorescence staining. Briefly, after macrophages were treated with LPS and AuNPs, the cells were incubated with Fura-4 AM (MCE, USA) and counterstained with DAPI (Beyotime). Fluorescence images were captured using a ZEISS 780 laser confocal microscope (Zeiss, Germany).