Liver samples were embedded in TissueTek OCT compound (Electron Microscopy Sciences, Japan). Five micrometers of the frozen section were fixed in 4% paraformaldehyde and blocked with 2% bovine serum albumin, then incubated with anti-mouse F4/80- eFluor® 570, or anti-mouse CD11b Alexa Fluor® 488 (eBioscience, San Diego, CA) for 2 hours at room temperature. After washing the sections were covered with Vectashield mounting medium containing DAPI and observed under a microscope (Leica M165 FC). In a blinded fashion, liver sections were first observed under low power (40×), 10–20 representative areas were selected and positive cells were counted under high power (×400). Quantitation was based on average numbers of positive cells/high power field. 3 mice/group/time point were analyzed.
To stain LSECs, liver sections (5μm) were stained with rat anti-mouse CD31 antibody (BD Biosciences, San Jose, CA) for 2 hours at room temperature, followed by FITC -labeled goat anti-rat IgG (Vector, Burlingame, CA). DAPI (blue) was used for nuclear counterstaining.
To stain LSECs, liver sections (5μm) were stained with rat anti-mouse CD31 antibody (BD Biosciences, San Jose, CA) for 2 hours at room temperature, followed by FITC -labeled goat anti-rat IgG (Vector, Burlingame, CA). DAPI (blue) was used for nuclear counterstaining.