Immunohistochemical and Immunofluorescence Analysis of Liver Tissue

For IHC, the liver tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned (4-μm thickness). They were then dewaxed, hydrated for 30 min at room temperature, and incubated overnight at 4 °C with the following primary antibodies: anti-CD31 (1:1000, Proteintech, Wuhan, China); anti-CD206 (1:10000, Proteintech, Wuhan, China), anti-HECTD3 (1:400, BIOS, Beijing, China). Subsequently, the slides were incubated with a horseradish peroxidase-labeled secondary antibody, and the immunoreactivity was visualized with 3,3-diaminobenzidine tetrahydrochloride (DAB). Tissue sections were counterstained with hematoxylin and visualized using Leica Microsystems at 200 × and 400 × magnification. Image-pro plus 6.0 was used for image analysis (Media CybernetiHOPE, Inc., Rockville, MD, USA).
IF was performed as previously reported^{44 (link)}. Specifically, the sections or cell slides were incubated with anti-CD31, anti-CD206, anti-TRAF3 (1:100, Proteintech, Wuhan, China), anti-Ly6G (1:100, Novus, Littleton, USA), anti-MPO (1:200, Servicebio, Wuhan, China), anti-CD11b (1:500, Servicebio, Wuhan, China) and anti-HECTD3 (1:100, BIOS, Beijing, China) antibodies. After washing, the slides were incubated with Alexa Fluor 594-conjugated secondary antibodies or Alexa Fluor 488-conjugated secondary antibodies (1:200, Proteintech, Wuhan, China).