Lillie mayer s hematoxylin

Liver sections were fixed in Boin’s solution or in 10% buffered formalin. The fixed liver sections were paraffin embedded and cut into sections for histological analysis. The sections were stained with Lillie-Mayer’s Hematoxylin (Muto Pure Chemicals Co, Japan) and eosin solution (Wako Pure Chemical Industries, Japan). NAFLD activity score (NAS) was calculated according to the criteria of Kleiner et al. [27 (link)]. To study collagen accumulation, the sections were stained with Picrosirius Red (PSR, Waldeck, Germany) and examined in bright field images at a magnification of 200× using a digital camera (DFC295, Leica, Germany) and ImageJ software (National Institute of Health, USA). Hepatic steatosis was assessed using frozen liver sections (10 μm) stained with Oil Red O dye using the Oil Red O staining (Abcam, Cambridge, MA, USA). To assess the extent of steatosis, the percent of liver tissue area covered by oil-red positive lipid droplets was calculated as described previously [28 (link)]. The percentage area positive for lipid (red color) was calculated from the mean of 20 fields (200×) for each animal using Nikon NIS Elements Software (Nikon Instruments Inc., Melville, NY, USA). All histological analysis was carried out by at least two blinded observers.