Immunofluorescence

Immunofluorescent staining for F4/80 was carried out as described in our previous study [22]. Briefly, frozen liver sections were rinsed in phosphate-buffered saline (PBS) for 5 min 3 times. Then, the sections were incubated with DAKO protein block (S3022, DAKO, Denmark) for 30 min at RT. Sections were covered with anti-F4/80 at a 1:200 dilution (eBioscience, 14-4801-82, USA) at 4 °C overnight. Sections were washed 3 times with PBS for 5 min and incubated with goat anti-rat secondary antibody (Invitrogen, A11077, USA) at a 1:200 dilution for 60 min at RT. Sections were rinsed 3 times with PBS for 2 min each time and mounted with DAKO fluorescent mounting media (S3023, DAKO).