B‐cell lymphoma 2 (BCL2) Immunohistochemical staining

Paraffin‐embedded liver sections were deparaffinized and rehydrated. To block endogenous peroxidase activity, sections were incubated with 5% H₂O₂ for 10 min at room temperature. After rinsing the slides with phosphate‐buffered saline (PBS), they were incubated with primary antibodies against BCL2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Biotinylated secondary antibodies (DakoCytomation, Carpinteria, CA, USA) directed against immunoglobulin were then added and incubated for 15 min. Protein expression level was evaluated using a streptavidin–biotin–peroxidase kit. Sections were stained with diaminobenzidine (DAB) solution as a chromogen for BCL2 detection and then counterstained with haematoxylin (Xiaohui et al. 2007). A negative control was generated using normal tissues but omitting the primary antibody. Slides were examined under a light microscope, and representative images were taken. The number of BCL2‐positive hepatocytes was counted morphometrically in five randomly selected high‐power microscopic fields within total six liver sections from ten rats/group using a computerized image system composed of a Leica Qwin 500 image analyser (England) which is connected to a Leica microscope and was expressed as cell number per μm² (Salama et al. 2013).