Mouse monoclonal anti 8 ohdg antibody

The kidneys were fixed in 10% formaldehyde overnight at 4 °C and processed for paraffin-embedding following standard procedures. Sections were cut at 3-μm thicknesses. For immunohistochemical analysis, some tissue sections were subjected to antigen retrieval by microwaving for 10 or 15 min in 10 mM sodium citrate buffer [pH 6.0]. Endogenous peroxidase activity was blocked by incubation with 3% hydrogen peroxide for 10 min. After PBS washing, sections were incubated with 1.5% normal goat serum for 20 min, followed by incubation with mouse monoclonal anti-8-OHdG antibody (1:50; Santa Cruz Biotechnology Inc., sc-66036) overnight at 4 °C. After three washes with PBS, the samples were incubated with biotin-conjugated goat anti-mouse IgG for 30 min at room temperature. After washing in PBS, the sections were incubated with streptavidin-conjugated peroxidase 30 min at room temperature. After PBS washing, the sections were incubated with DAB followed by examination under the microscope.

The immunohistochemistry staining methods were performed as previously described35 (link). After dewaxing, antigen retrieval and blocking of endogenous peroxidase, the sections were fixed with goat serum and incubated with a 1:1000 dilution of rabbit monoclonal anti-CGL antibody (Abcam, Cambridge MA, USA), a 1:2000 dilution of rabbit monoclonal anti-CBS antibody (Abcam, Cambridge MA, USA), and a 1:20 dilution of mouse monoclonal anti-8-OHdG antibody (Santa cruz biotechnology, CA, USA) overnight at 4 °C, followed by incubation with biotin-conjugated goat anti-rabbit IgG (Invitrogen, Carlsbad, CA, USA) and streptavidin- conjugated peroxidase (Invitrogen, Carlsbad, CA, USA). Finally, sections were detected under a microscope after reaction using 3,3′-diaminobenzidine (DAB) (Invitrogen, Carlsbad, CA, USA). The intensity of immunohistochemical staining of 8-OHdG was analyzed in ten random fields (×400) of renal cortex per rat using Image J software (NIH, MD, USA).