Hrp conjugated anti mouse igg

Formalin-fixed, paraffin-embedded tissue sections from normal ovary tissue and serous tumors were cut into 4–6-μm sections and transferred to slides. Samples were deparaffinized in xylene and rehydrated via an ethanol gradient (100%, 95% ×2, 85%, 75% ×2, 50%, and distilled water). Slides were then washed, endogenous peroxidase activity was blocked by incubation in 3% H₂O₂, and samples were preincubated with 10% goat serum, and then with monoclonal mouse anti-PDCD5 antibody (1:1000) for 1 h at room temperature in a humid chamber. Secondary staining with HRP-conjugated anti-mouse IgG (Zhongshan Golden Bridge Biotechnology, Beijing, China) was performed using a DAB peroxidase substrate kit (Zhongshan Golden Bridge Biotechnology, Beijing, China). Nuclei were counterstained with hematoxylin. In negative controls, the primary antibody was replaced with an isotype control. All staining was evaluated using the Leica Q550CW software system (Germany) and Qwin image analysis software. We randomly selected five independent fields from each slide; the average density of positive cells these five fields was calculated as the PDCD5 expression level.