Immunofluorescence staining

Liver tissue sections (5 µm) were fixed with 4% paraformaldehyde (PFA), submerged in 30% sucrose overnight, frozen in Tissue-Tek Optimal Cutting Temperature (OCT) compound and immunostained with primary antibodies against CD8α, Ki-67, c-Met, α-SMA and Fas (working dilution 1:400) in DMEM/F12 containing 5% normal goat serum (NGS) at 4 °C overnight for purpose target detection. A fluorescent dye (Alexa Fluor 488 or Cy3)-conjugated secondary antibody (working dilution 1:2000–4000) diluted in DMEM with 5% NGS was then added, and the sections were incubated at room temperature (RT) for 1 h and then mounted in Vectashield with 4’,6-diamidino-2-phenylindole (DAPI) to label the nuclei. The samples were rinsed again with phosphate-buffered saline (PBS) and ddH₂O before mounting. The proportions of these stained cells in defined regions were calculated and analyzed. Three to 6 different animals were assayed with at least 16 sections/animal. Sample sizes were determined based on power estimates. All images were taken by an investigator blinded to the treatment of the individual animals. For quantification of the proportion of specific stained and unspecific stained cells was taken from multiple sections at least 2 different liver tissue regions from 3 to 6 animals.