Immunohistochemical Analysis of Liver Fibrosis

Paraffin-embedded liver sections were deparaffinized with xylene and dehydrated in gradually decreasing concentrations of ethanol. Immunohistochemical staining was performed according to the described procedure [6 (link),38 (link)]. For immunohistochemical staining, the liver sections were incubated with a primary antibody (1:100 dilution) for 1 h at 37 °C. Primary antibodies used were as follows: antifibronection (BD Biosciences, San Jose, CA, USA) and anti-α-SMA (A2547, Sigma-Aldrich, St. Louis, MO, USA). After 3 serial washes with PBS, the sections were processed by an indirect immunoperoxidase technique using a commercial Envision System kit (DAKO, Carpinteria, CA, USA). Immunohistochemical images were viewed with an Eclipse 80i microscope (Nikon, Tokyo, Japan).