Lieca qwin 500 image analyzer

The immune-histochemical analysis of AR and PCNA were done according to Abdel-Rahman et al. (2017) . The tissue specimens were deparaffinized and rehydrated then pretreated with citrate buffer pH 6 for 20 min for antigen retrieval. The sections were incubated with primary antibodies of rabbit polyclonal anti-androgen receptor antibody (ab133273; Abcam, Cambridge, UK) diluted 1:500 and Rabbit polyoclonal anti-PCNA antibody (ab18197; Abcam, Cambridge, UK) diluted 1:4000 for three hours in a humidified chamber. The sections were incubated with secondary (HRP) antibody (ab205718; Abcam, Cambridge, UK). DAB (Sigma) was used as chromogen to visualized reaction. Finally, the slides were counterstained with Mayer hematoxylin and mounted with DPX. The images were analyzed by Lieca Qwin 500 Image Analyzer (Leica, Cambridge, England). In each group, five sections were examined. Color density of the immunopositive cells (dark brown) was evaluated.

The immunohistochemical analysis of the insulin, glucagon and somatostatin content of the pancreatic islets were performed according to the methods described by Jevdjovic et al (Jevdjovic et al., 2005 (link)). After deparaffinization, rehydration, blocking of the endogenous peroxidase activity and antigenic retrieval, the tissue sections were incubated for 3 h with mouse monoclonal anti-insulin (18–0066; Zymed, San Francisco, CA) at a dilution of 1:50, sheep polyclonal anti-glucagon antibody (ab36232; Cambridge, UK) at a dilution of 1:100 and rabbit polyclonal anti-somatostatin antibody (ab108456; Cambridge, UK) at a dilution of 1:50. The tissue sections were incubated with a biotinylated goat anti rabbit antibody (Thermo scientific, USA) and rabbit anti-sheep antibody (ab6747; Cambridge, UK) for 10 min. The sections were incubated finally with Streptavidin peroxidase (Thermo scientific, USA), 3,3′-diaminobenzidine tetrahydrochloride (DAB, Sigma) and counterstained with haematoxylin. In each field, the immunopositive areas were analyzed by Lieca Qwin 500 Image Analyzer (Leica, Cambridge, England) in 10 microscopic fields under high-power field (X400) microscope. Percentage of positive stained area (%) was calculated as mean of 10 fields/slide.