4.4. Hematoxylin and Eosin (H&E) Staining and Liver Injury Score Analysis

H&E staining for liver histological change was observed in the same manner as described above [19,40]. The slides were soaked in Mayer’s hematoxylin (DAKO, Glostrup, Denmark) for 30 s, washed with water until clean, and again the slides were soaked in eosin (Sigma-Aldrich Co., St. Louis, MO, USA) for 10 s and then washed with water. After air drying, the slides were left at room temperature, then soaked twice each in turn in 95% ethanol, 100% ethanol, 50% ethanol with 50% xylene solution and then 100% xylene. After air drying at room temperature overnight, the slides were mounted on coverslips using Permount^® (Thermo Fisher Scientific, New Jersey, NJ, USA).

Histopathological score of liver was evaluated in the same manner as described above [40]. Images from the H&E-stained slides were captured using an Image-Pro^® plus computer-assisted image analysis system (Media Cybernetics Inc., Silver Spring, MD, USA) and images were observed with an optical microscope (Olympus, Tokyo, Japan). The images of the slides were evaluated by a blind test of pathologists. The histological score was used to grade the severity of the necrosis and inflammatory process [41,42], as shown in Table 1. The score was calculated by summing all the grades of each item.

Histological scoring parameters.