Anti p erk1 2 9101

Antigen retrieval was performed using heat-induced epitope retrieval buffer at 95°C for 20 minutes. The sections were blocked in PBS containing 0.25% Triton-X and 10% FBS for 30 minutes at room temperature. Primary antibodies, anti-PCNA (MA5-11358) or anti-pERK1/2 (9101), both purchased from Cell Signaling Technology, were used at a dilution of 1:500 in 10% FBS in PBS solution for overnight incubation. After washing with PBS, the slides were incubated for 1 hour with respective secondary antibodies anti-mouse Alexa Fluor 647 (A31571) or anti-rabbit Alexa Fluor 488 (A11034), purchased from Invitrogen, at dilution of 1:500. Kidney sections were then counterstained with the nuclei marker, 4′,6diamidino-2-phenylindole (DAPI) for 10 minutes, and covered with glass coverslip using antifading solution and sealed. Slides were imaged using a fluorescent microscope (Olympus BX51; Olympus America, Center Valley, PA). Each experimental group had six mice, and three kidney sections were analyzed from each experimental mouse. Five square areas from each kidney section, covering more than 80% of the total kidney area, were imaged and used for quantification of MFI using ImageJ software. The nuclei number was analyzed from each image using "Analyze Particle" mode. The ratio of MFI to the number of nuclei was used for each image for statistical analysis.