The animals that underwent cell transplantation were euthanized 30 days after surgery. Livers were removed and immediately cut into 5-mm-thick slices. Liver sections were fixed in 4% paraformaldehyde (Merck KGaA, Darmstadt, Germany) in PBS. Some sections were embedded in Tissue-Tek (Sakura Finetechnical Co., Tokyo, Japan), frozen using isopentane/dry ice, and kept at −80 °C until use. The frozen sections were used for enzyme-based histochemical or immunohistochemical examinations.
Frozen liver sections with a thickness of 7 μm were prepared and air-dried. We used the ABC method for immunohistochemistry. Deparaffinized sections were treated with 0.6% H2O2 in methanol for 30 min to suppress endogenous peroxidase activity. After blocking with BlockAce for at least 30 min at 4 °C, the slices were incubated with the primary antibody (Supplemental Table S1 ) for 60 min, rinsed with PBS, and then incubated with a secondary antibody for 30 min. Next, the sections were incubated with an avidin-conjugated biotinylated antibody (VECTASTAIN ABC kit; Funakoshi, Osaka, Japan). 3,3′-diaminobenzidine tetrahydrochloride (DAB) was used as a substrate. Nuclei were counterstained with hematoxylin. Images were obtained using a Zeiss 780 confocal microscope (Carl Zeiss, Oberkochen, Germany) and a confocal laser microscope (Olympus, Tokyo, Japan).
Frozen liver sections with a thickness of 7 μm were prepared and air-dried. We used the ABC method for immunohistochemistry. Deparaffinized sections were treated with 0.6% H2O2 in methanol for 30 min to suppress endogenous peroxidase activity. After blocking with BlockAce for at least 30 min at 4 °C, the slices were incubated with the primary antibody (