Formalin-fixed liver samples were processed and 5 μm thick paraffin sections were used for F4/80 immunohistochemistry. Briefly, de-paraffinized liver sections were treated with 3% hydrogen peroxide followed by antigen retrieval. The sections were blocked with 10% horse serum, and incubated with primary antibody, which was then linked to the biotinylated secondary antibody followed by avidin-biotin complex (ABC Vectastain kit; Vector Laboratories, Burlingame, CA, USA), and diaminobenzidine, which gave a brown reaction product. The sections were then counterstained with hematoxylin. F4/80 expression levels were identified by the increased brown colored staining versus normal controls by using the NIH Image analysis software.
