Alexa 594

According to previously published methods, the expression of M1 and M2 markers in BV2 microglia were detected using immunocytochemistry (ICC).^{28,29 (link)} BV2 microglia cells were grown on 24-well plate with a glass slide chamber for overnight and were treated with siTLR4/M3 and siCtrl/M3 complexes for 4 h, followed by OGD treatment. After incubating the cells in normal cell culturing condition for 24 h, and fixed with 4% PFA in 0.1 M PBS. Fixed cells were rinsed using PBS, permeabilized with 0.1% Triton-X 100 for 10 min, and then incubated with 2% BSA in PBS for 1 h to block nonspecific binding. Then the cells were probed with primary antibodies for the M1 phenotype (iNOS, 1 : 400; Cell Signaling Technology, MA, USA) or the M2 phenotype (CD206, 1 : 400; Cell Signaling Technology, MA, USA). For visualization, the secondary antibodies, anti-rabbit Alexa-594 (1 : 1000; Cell Signaling Technology, MA, USA), or goat anti-rabbit IgG-FITC (1 : 1000; Abcam, Cambridge, MA) were used along with the nuclear marker DAPI (1 : 1000; Thermo Scientific, MA, USA). Images were obtained by a Nikon fluorescence confocal microscope (Nikon, C2⁺).

Liver specimens were fixed in 4% paraformaldehyde for at least 24 h and then embedded in paraffin. Sections (4 μm thick) were cut for H&E, Sirius Red staining, and Perls’ Prussian Blue staining.
For immunohistochemistry, formalin-fixed paraffin-embedded sections were deparaffinised and used to assess α-smooth muscle actin (α-SMA) (1:400, cat# 19245, Cell Signaling Technology, MA, USA), F4/80 (1:200, cat# 70076, Cell Signaling Technology), 8-OHdG (1:200, cat# sc-66036, Santa Cruz, USA) and Ki67 (1:400, cat# 12202, Cell Signaling Technology). Images were acquired using a microscope (Nikon Ci-E).
For immunofluorescence, liver cryosections were deposited on glass slides. After blocking with PBS containing 1% bovine serum albumin and 0.2% Triton X-100, the sections were incubated with primary antibodies, including anti-F4/80 (1:200, cat# ab6640 Abcam, USA), anti-STING (1:200, cat# 19851-1-AP, Proteintech), anti-CD68 (1:200, cat# 26042, Cell Signaling Technology), anti-iNOS (1:200, cat# 18985-1-AP, Proteintech), and anti-CD206 (1:200, cat# 18704-1-AP, Proteintech) at 4 °C overnight. The secondary antibodies were conjugated with Alexa 488 (1:200, cat# 4408, or # 4412, Cell Signaling Technology) or Alexa 594 (1:200, cat# 8889, Cell Signaling Technology), and the nuclei were counterstained with DAPI.