Anti e selectin

The cryosections of the aortic root were fixed in 4% paraformaldehyde and permeabilized using 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA). The sections were blocked using 5% normal goat or donkey serum (Abcam) at room temperature for 2 h and then incubated with primary antibodies, including anti-CD68 (1:100, Abcam, ab31630), anti-VCAM-1 (1:100, Abcam, ab134047), anti-E-Selectin (1:100, Santa Cruz, sc-137054), overnight at 4 °C followed by appropriate fluorescence-conjugated secondary antibodies (ThermoFisher, Waltham, MA, USA) for 2 h at room temperature in the dark. Nuclei were stained with Hoechst 33342 (ThermoFisher, Waltham, MA, USA) and mounted with a fluorescence mounting medium (Electron Microscopy Sciences, Cat#17985-10, Hatfield, PA, USA). Images were acquired using an Olympus FV1200 confocal microscope (Olympus Corporation, Tokyo, Japan). Quantification of immunofluorescence staining was performed with the Image J software.

Paraffin-embedded liver tissue from DIO, DIO+BDCM (1w), Lep KO (1w), Lepr KO (1w), MCS (4w), MCD (4w), miR21 KO+BDCM (1w) and miR21 KO+MCD (4w) groups was cut into 5 μm thick sections. Each section was deparaffinized using standard protocol. Briefly, sections were incubated with xylene twice for 3 min, washed with xylene:ethanol (1:1) for 3 min and rehydrated through a series of ethanol (twice with 100%, 95%, 70%, 50%), twice with distilled water and finally rinsed twice with phosphate buffered saline (PBS). Epitope retrieval of deparaffinized sections was carried out using epitope retrieval solution and steamer (IHC-world, Woodstock, MD) following manufacturer’s protocol. The anti-mouse primary antibodies (i) anti-VEGFR-2 was purchased from AbCam Inc. (Cambridge, MA), (ii) anti-ICAM-1, and (iii) anti-E-selectin were purchased from Santa Cruz biotechnology, Inc. (Santa Cruz, CA), and used in 1:150 dilutions. Species-specific anti-IgG secondary antibody conjugated with Alexa Fluor 633 (Invitrogen, California, USA) was used to localize the sinusoidal endothelial dysfunction biomarker proteins. Sections were mounted in ProLong gold antifade reagent with DAPI. Images were taken under 20× objectives using Olympus BX51 microscope.