Bcl 2

After euthanasia, the penises were harvested. The midshaft segments were fixed in 10% formalin and embedded in paraffin. Paraffin-embedded specimens were stained with Bax (1:250, Abcam) and Bcl-2 (1:500, Abcam) antibodies. Then, the sections were washed and incubated with secondary antibodies (1:100 dilution, Zhongshan Golden Bridge Biotechnology Co., Beijing, China). Thereafter, the sections were incubated with 3,3-diaminobenzidine, and the cell nuclei were stained with hematoxylin. Primary antibodies were replaced with normal serum from the host species and incubated with the secondary antibody as a negative control. Sections were examined under a light microscope. Slides were examined by an observer blinded to the treatment group. Computerized densitometric analyses of Bax and Bcl-2 expression in cavernous tissue in the images were performed using Image-Pro Plus version 5.0 software (Media Cybernetics Inc., Bethesda, MD, USA).

Immunohistochemical staining was performed to detect the expression of Bax and Bcl-2 in paraffin-embedded tissue sections of rat ovaries using a standard protocol of horseradish peroxidase (HRP)-conjugated rabbit anti-human IgG described previously (17 (link)). The whole tissue slides from the selected cohort were stained with an automated procedure. The primary antibodies anti-Bax and anti-Bcl-2 (Cell Signaling Technology, Danvers, MA, USA) were manually applied at 1:1,000 and 1:2,000 dilutions, and the slides were incubated at 37°C for 1 h. Amplification and detection were performed using the UltraView Amplification and DAB detection kits. The slides were counterstained with hematoxylin for 4 min and post-counterstained with bluing agent for 4 min. The slides were then washed with mild detergent and dehydrated in a series of 70% to 100% alcohol baths, cleared in a xylene bath, and cover slipped to analyze the average integrated optical density (IOD) of Bax and Bcl-2 staining using Image-pro plus 6.0 software (Media Cybernetics, USA).