4.4. Sirius Red Staining and α-Smooth Muscle Actin Immunohistochemistry

Hepatic fibrosis was assessed in paraffin embedded liver sections (2–3 µm) stained with 0.1% Sirius Red in saturated picric acid (Chroma, Münster, Germany). Immunohistochemistry against α-smooth muscle actin (α-SMA) was performed in snap frozen liver samples. Cryosections (5–10 µm) of liver samples were fixed and incubated with mouse anti-α-SMA antibody (Sigma Aldrich, Munich, Germany) and a biotinylated donkey anti rat secondary antibody (Abcam, Cambridge, UK). Stained sections were digitalized using Pannoramic MIDI (3DHISTECH, Budapest, Hungary) and positive staining was quantified using Histoquant (3DHISTECH, Budapest, Hungary), as previously described [13,44].