Biozero bz 700

Tissue samples were fixed with periodate-lysine-paraformaldehyde fixative at room temperature for 3 h. Following cryoprotection with 30% sucrose prepared in 0.1 M phosphate buffer (pH 7.2), fixed liver samples were embedded in Tissue-Tek O.C.T. compound (Sakura Finetek USA, Torrance, CA, USA) and frozen at –80°C. Samples were cut into 8 μm sections using a cryostat and incubated for 1 h at room temperature with Dako Protein Block Serum-Free solution (Glostrup, Denmark) to block any nonspecific binding. The sections were then incubated overnight at 4°C with a rat anti-CD1d monoclonal antibody (BD Bioscience, San Jose, CA, USA) and a rabbit anti-F4/80 polyclonal antibody (Cell Signaling Technology, Beverly, MA, USA). After washing three times in PBS, the sections were incubated for 1 h at room temperature with the following secondary antibodies: Alexa Fluor 488-conjugated donkey anti-rabbit IgG and Alexa Fluor 594-conjugated donkey anti-rat IgG (Invitrogen, Carlsbad, CA, USA). Images of the stained sections were captured using a fluorescence microscope (Biozero BZ-700; Keyence).