Horseradish peroxidase hrp conjugated goat anti rabbit or rat anti mouse secondary antibody

Renal tissue sections were subjected to immunohistochemical staining for F4/80 and Fi67. For immunohistochemical staining, 3-mm renal sections were deparaffinized and rehydrated in a graded alcohol series. The sections were immersed in 3% hydrogen peroxide for 10 min to block endogenous peroxidase activity and then incubated in buffered normal horse serum to block nonspecific binding. Before immunochemistry, sections were subjected to antigen retrieval by immersion in 0.1 mol/L citrate buffer (pH 6.0) for 25 min, followed by heating in an electrical pressure cooker for 5 min. Sections were incubated with rabbit polyclonal anti-F4/80 (1:100; eBioscience, San Diego, Calif) and mouse monoclonal anti-CDFi67 (1:100; Abcam) primary antibodies overnight at 4°C. Control experiments omitted either the primary or secondary antibody. On the next day, sections were incubated with a horseradish peroxidase (HRP)-conjugated goat antirabbit or rat anti-mouse secondary antibody (Beijing Zhongshan Biotechnology Co., Beijing, China) for 1 h at room temperature. Then, 3,3-diaminobenzidine tetrahydrochloride (DAB; Beijing Zhongshan Biotechnology Co., Beijing, China) was applied to the slides to develop a brown color. Counterstaining was performed with hematoxylin, and photomicrographs were captured with an Olympus camera.