Mouse monoclonal anti pcna antibody

To assess the relation of megalin expression to the presence of proliferation marker PCNA in tumor tissue, double immunofluorescence staining was carried out on 4μm paraffin-embedded OSCC tissue sections. Briefly, following deparaffinization, rehydration and heat-mediated epitope retrieval, slides were incubated with a blocking solution (1% BSA and 0.001% NaN3 in PBS) for 1 h at room temperature. Immediately afterward, rabbit polyclonal anti-megalin antibody (H-245, Santa 198 Cruz Biotechnology, Dallas, TX, USA; diluted 1:50 in blocking solution) and mouse monoclonal anti-PCNA antibody (Abcam, UK; diluted 1:100 in blocking solution) were added, and slides were hereafter incubated for 12 h at 4 °C in a humid chamber. Tissue sections were then rinsed thrice in PBS being subsequently incubated with Alexa Fluor 488- conjugated donkey anti-rabbit secondary antibody (Thermo Fisher Scientific, Waltham, MA, USA; diluted 1:300 in blocking solution) and Alexa Fluor 555-conjugated goat anti-mouse secondary antibody (Thermo Fisher Scientific, USA; diluted 1:500 in blocking solution) for 1 h at room temperature and in a humid and dark environment. Nuclei were visualized using 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; Thermo Fisher Scientific, USA; diluted 1:1000 in PBS). Analysis and image capturing were performed using an Olympus BX51 microscope and DP50 camera (Olympus, Japan).

The paraffin-embedded tumor tissue sections were dewaxed, rehydrated and treated with antigen retrieval in 10 mM citrate buffer (pH 6.0) for 15 min in a microwave-oven. Sections were immersed in 3% hydrogen peroxide solution for 10 min to quench endogenous peroxidase activity. Non-specific binding was prevented by incubation with 5% normal goat serum for 15 min. The sections were incubated with mouse monoclonal anti-PCNA antibody (Abcam, Cambridge, UK), rabbit monoclonal anti-CD34 antibody (Abcam, Cambridge, UK), or anti-vascular endothelial growth factor (VEGF) antibody (Abcam, Cambridge, UK) overnight at 4 °C. Then, antibody binding was detected using a horseradish peroxidase-conjugated secondary antibody (Zhongshan Golden Bridge Biotechnology Co., Beijing, China) for 1 h at 37 °C. The sections were visualized with diaminobenzidine (DAB) solution, lightly counterstained with hematoxylin, and observed using light microscopy.