Picric acid fast green

Double fluorescence staining was performed with liver sections as we described previously 9. Briefly, paraformaldehyde‐fixed liver sections were blocked with normal serum and incubated with primary antibody against 4‐hydroxynonenal (4‐HNE, 1:50; Abcam, Cambrige, MA, USA), Sonic hedgehog (Shh, 1:50; Santa Cruz), Ser 473 phosphorylated AKT (p‐AKT, 1:100; Cell Signaling Technology Inc, Bevely, MA, USA), β‐catenin (1:100; Santa Cruz), PPARγ (1:100, Abcam), GATA3 (1:50; Santa Cruz) and primary antibody against synaptophysin (SYP, 1:10; Abcam), a marker for quiescent and activated HSCs 21, followed by incubation with DyLight594‐conjugated secondary antibody (1:500; ImmunoReagents, Inc, Raleigh, NC, USA) and DyLight488‐conjugated secondary antibody (1:500; ImmunoReagents, Inc). The images were captured with the fluorescence microscope and representative images were shown.
Sirius red was used to stain collagen on liver. Briefly, mouse liver sections were stained with picric acid‐fast green (Amresco, Solon, OH, USA) and then incubated with picric acid–sirius red (Amresco) for 1 hr. The images were captured with light microscope and representative images were shown (Data S2).

Double fluorescence staining was conducted for examining the respective proteins in HSCs in mouse livers. Briefly, the livers were fixed with 4% buffered paraformaldehyde. After the liver sections were blocked with normal serum, the samples were incubated with primary antibody and the respective secondary antibody (shown in Supporting information). The nuclei were stained by Hoechst 33342 (Sigma). The images were captured with the fluorescence microscope. The positive cells were counted in six randomly chosen high‐power fields at 200‐fold magnification.
For evaluating liver fibrosis, Sirius red was used to stain collagen in liver sections. Briefly, the samples were stained with picric acid‐fast green (Amresco, Solon, OH, USA) for 10 minutes and then incubated with picric acid–Sirius red (Amresco) for 1 hour. Images were captured with light microscope. The stained areas were quantified using ImageJ software (NIH, Bethesda, MD, USA).