Histopathological and immunohistochemical investigations of Bax, caspase-3, and Bcl2

The formalin-fixed liver specimens were rinsed, dehydrated in escalating degrees of ethyl alcohol, clarified in xylene, and further processed for the paraffin technique (Layton et al. 2018). A microtome (Leica RM 2155, England) was used to slice three successive paraffin sections with a thickness of five microns. Before the microscopical examination, the sections were stained with hematoxylin and eosin as per protocol, mounted in DPX, and covered with a glass slide (Suvarna and Layton 2013).

For immunohistochemical workup, the previously obtained tissue, ten slides per biomarker per group (5 µm paraffin sections) were managed for immunohistochemical staining following the ABC technique described by Hsu et al. (1981) using the following primary antibodies: (a) for caspase-3, rabbit monoclonal (EPR18297) to anti-mice caspase-3 (Abcam, Cat. no. ab184787, dilution 1;1000); (b) for Bax, rabbit polyclonal anti-Bax antibody (Abcam, Cat. no. ab53154, dilution 1; 50); (c) for BcL-2, rabbit monoclonal (EP10625) to anti-mice BcL-2 (Abcam, cat. no. ab203516, dilution 1;500). (ABCAM Inc., Cambridge, UK). Also, negative sections from the control were obtained by incubating with phosphate buffer saline to replace the primary antibodies.

To measure positive reactivity, images of different sections stained with antibodies were examined under a microscope powered by an Olympus BX-50 in Tokyo, Japan, with a 1/2 × photo adapter and a 40 × objective. The images were captured using an Olympus LC20 digital camera, which was put on an Olympus microscope. The images were analyzed using a computer with an Intel® Core I3® and the Russian program Video Test Morphology 5.2, which has a dedicated method for immunohistochemical analysis and stain quantification. The system determined caspase-3, Bax, and Bcl2 expression percentages in a certain region. For quantitative analysis, we selected five representative areas in total with both positive cell areas and areas without expression. If a tissue section had areas with both low abundance and high abundance of stained cells, both areas were selected as representative areas and included in the analysis. Individual cells were identified by strong brown stain and Image analysis software (JID801D) assessed positive cells. We counted the number of positive expressed cells per mm² and converted them into area %. The cell counting was repeated three times for each area. All images were analyzed in a blinded fashion.