Sc 66036

Primary monoclonal antibody, mouse anti-proliferating cell nuclear antigen (PCNA) (1:100, M0879, Dako, Glostrup, Denmark) was used as a proliferation marker, incubated overnight at 4 °C with the negative control reagent (V1617; Dako, Glostrup, Denmark). Next, LSAB2 System-HRP visualization reagent for use on rats (K0609; Dako, Glostrup, Denmark) was applied according to the manufacturer’s instructions. Antibodies to oxidative/nitrosative stress markers were anti-8-OHdG (1:300, sc-66036) and anti-nitrotyrosine (1:100, sc-32757), both from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). After overnight incubation at 4 °C, sections were treated with Dako REAL EnVision/HRP, Rabbit/Mouse reagent (K5007, Dako, Agilent Technologies Inc., Santa Clara, CA, USA); 3,3′-diaminobenzidine-tetrahydrochloride (DAB) was used for signal staining and hematoxylin for counterstaining. Immunohistochemistry was done on at least 6 samples from different groups of treated animals.

Five 4-µm cross-sections, mounted on previously silanized slides, were
used to show the expression of 8-OHdG, MMP-2 and MMP-9. The slides were then
deparaffinized, cleared, hydrated and washed in running water. Then, endogenous
peroxidase activity was blocked with 0.3% hydrogen peroxide, protein blocking
was performed with 0.3% skim milk diluted in PBS and the slides were incubated
overnight with anti-8-OHdG (SC66036 Santa Cruz® Biotechnology, CA, USA)
primary antibody, titrated 1:100, and MMP2/72KDa and MMP9/KDa (SC-10436 Santa
Cruz® Biotechnology, CA, USA; SC-6840 Santa Cruz^®Biotechnology, CA, USA), titrated 1:150 in PBS-BSA 0.1%. All slides were then
placed in a humid chamber at 4°C overnight. The material was washed with PBS
buffer and incubated with biotinylated secondary antibody. For revelation, the
3-3´diaminobenzidine chromogenic substrate was used at a ratio of 0.06 g per 100
mL of PBS, and 1 mL of 20-volume H₂O₂ for five minutes at
37°C and counter-stained with Mayer’s hematoxylin for 3 minutes. Finally, the
slides were mounted with cover slips and entellan® for analysis under
light microscopy.

Intracellular ROS production in the chondrocytes were detected with the cell-permeable fluorescent dye dihydroethidium (DHE), and oxidative DNA damage was determined with 8-oxo-dG antibody, the oxidized derivative of deoxyguanosine. The primary chondrocytes were treated with IL-1β (10 ng/mL) with or without 2-deoxy-d-glucose (2-DG; 2 mM), IACS-010759 (2 µM), 1400W (10 µM), and APX-115 (10 µM) for 24 h. The cells were fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.25% Triton X-100, and washed with phosphate-buffered saline (PBS) three times. To assess intracellular ROS production, 5 µM DHE was added for 30 min at 37 °C. The cells were washed with PBS, and photos of the cells were taken with an KI-3000F fluorescence microscope (Korealabtech, Pyeongtaek, Republic of Korea). For the immunofluorescence staining of 8-oxo-dG, the cells were incubated with primary antibody for 8-oxo-dG (sc-66036; Santa Cruz Technology, Beverly, MA, USA) overnight at 4 °C and incubated with a secondary antibody for 2 h and then photographed under the fluorescence microscope. Fluorescence-positive cells were quantified with ImageJ software (version 1.8.0, National Institutes of Health, Bethesda, MD, USA).