Alpha smooth muscle actin α sma

Mice liver tissues were fixed in 10% buffered formalin and embedded in paraffin. Liver sections were routinely stained Hematoxylin and Eosin and Sirius Red. Frozen sections from the different groups of mice were stained for Oil Red O. Diagnosis of NASH was based on the presence of steatosis, inflammation and hepatocyte ballooning. The severity was assessed by using the NAS scoring system [13] (link).
For Immunohistochemistry, liver sections were incubated with primary monoclonal antibodies against Alpha-Smooth Muscle Actin (αSMA) (DakoCytomation, Carpinteria, CA), p-AKT Ser473, β-Catenin (Cell Signaling, Boston, MA), p-c-Myc Ser373 (Biorbyt, Cambridge, UK) and Glypican-3 (Cell Marque, Rocklin, CA). Antibodies against Ki67 and F4/80 were from ABCAM, Cambridge, UK. In Situ Cell Death (TUNEL) kit was from Roche, Manneheim, Germany. Detection of positive staining was performed by using DAB reagent (Sigma-Aldrich, St. Louis, MO).
Quantitative morphometry was performed as previously described [14] , [15] (link).

Mice were perfused with PBS through left ventricle to flush blood, followed by 4%-PFA/PBS. Aorta was harvested and further fixed in 4% PFA/PBS at 4°C for 2–3 hours. Vessels were then incubated in a 15% sucrose/PBS solution at 4°C overnight, frozen with OCT compound, and cut 8–10 μm in each slice. After drying and removing OCT compound with PBS, 8–10 μm thick sections were incubated with primary antibodies, including von Willebrand factor (vWF) (1:100, DAKO, Carpinteria, CA, USA), CD68 (1:100, AbD), alpha-smooth muscle actin (α-SMA) (1:100, Dako), CD-31 (1:100, BD Biosciences, San Jose, CA, USA), Intracellular Adhesion Molecule-1 (ICAM-1) (1:100, Abcam) and Collagen typeI (1:100, Abcam), with 5% goat serum overnight at 4°C.
The following day, these sections were washed with PBS and incubated with secondary antibodies conjugated to Alexa Fluor 488 or 568 (1:200, Life Technologies) for 1 hour. Finally, vessels were washed with PBS, mounted with media containing DAPI (Invitrogen/Life Technologies), and evaluated using fluorescent microscope (Eclipse E800; Nikon).