Immunohistochemistry and X-gal staining

Sections from human liver specimens were processed by Masson trichrome staining for histologic evaluation. Immunostaining on human biopsies and murine livers was performed using the iVIEW DAB Detection Kit for the Ventana BenchMark XT–automated slide stainer. Primary antibodies for macroH2A1.1 and macroH2A1.2 were previously generated (3). Anti-5-Methylcytosine (33D3) antibody was obtained from Active Motif (cat. no. 39649); anti-GLB1/Beta-Galactosidase Antibody (clone 5H2) IHC-plus LS-{"type":"entrez-nucleotide","attrs":{"text":"B10217","term_id":"2091336","term_text":"B10217"}}B10217 was obtained from LSBio. macroH2A1.1, macroH2A1.2, and anti-GLB1 primary antibodies were diluted 1:100; anti-5-Methylcytosine primary antibody was diluted 1:1,000. Positive and negative controls were run concurrently. Means of triplicate counts were used for statistical analyses. For X-Gal staining of murine livers, frozen sections of 5-µm thickness were used and processed as previously described (30). Slides were counterstained with eosin, mounted with aqueous medium, and observed with an optical microscope (Nikon ECLIPSE Ni) connected to a digital camera (DS-Fi2). The percentage of blue staining, indicating β-galactosidase activity, was calculated in 10 random high-power field (HPF) at 400× magnification and expressed as means. Also, after performing quantitative analysis, the results were expressed in a semiquantitative scale (−: 0%; +: 1%–33%; ++: 34%–66%; +++: 67%–100%). All analyses were performed in triplicate by three independent pathologists.