8 hydroxy 2 deoxyguanosine 8 ohdg

Paraffin-embedded kidney sections were deparaffinised and incubated with primary antibodies against collagen IV (dilution 1:1000, Abcam Ltd, Cambridge, USA), fibronectin (dilution 1:500, Abcam), or 8-hydroxy-2'–deoxyguanosine (8-OHdg) (dilution 1:200, Cell Signaling Technology, Beverly, USA) at 4°C overnight, followed by horseradish peroxidase anti-rabbit Envision system (Dako Tokyo, Japan) the following day.
Alternatively, frozen sections were incubated in the widely used markers of murine macrophage populations, rat anti-mouse F4/80 monoclonal antibody (dilution 1:100, ABD Serotec, USA) or rat anti-mouse CD68 antibody (dilution 1:100, ABD Serotec) for one hour. Thereafter, they were incubated with a secondary HRP labelled goat anti-rat antibody for 30 min (ABD Serotec, dilution 1:200).
Antigen-antibody reactions were visualized with 3.3diaminobenzidine tetrahydrochloride (Dako) and counterstained. The tissue specimens were examined by light microscopy using the Olympus photomicroscope. For fibronectin, collagen IV, 8-OHdg, CD68, and F4/80, six consecutive non-overlapping fields from each section of renal cortex were photographed under high magnification. Stained areas were quantified using Image J software (NIH, UK).

Immunohistochemistry (IHC) staining was performed as previously described [8 (link)]. Briefly, tissues were fixed in 10% formalin for 36-h and embedded in paraffin or frozen-embedded in OCT solution (Tissue-Tek). Paraffin sections were prepared at a 4-μm thickness and mounted on microscope slides (Trajan Scientific and Medical, VIC, Australia). Antigen retrieval was performed at 99 °C for 20 min in 0.01 M, pH 6.0 citric buffer. Endogenous peroxidase was deactivated with 3% H2O2 (Sigma-Aldrich, Dublin, Ireland). The slides were then blocked by Protein Block Serum-Free (Dako, Glostrup, Denmark), and incubated with one of the primary antibodies, which included Fibronectin, Collagen type I (dilution 1:750, Abcam, Cambridge, UK), and 8-hydroxy-2′ -deoxyguanosine (8-OHdg, dilution 1:200, Cell Signalling Technology, MA, USA). After overnight incubation at 4 °C, biotinylated secondary anti-rabbit IgG antibodies (Dako) were incubated for 30 mins and finally horseradish peroxidase (HRP)-conjugated streptavidin (Dako) for 10 mins. Using a light microscope (Leica DM750 photomicroscope with ICC50W digital camera), six consecutive non-overlapping fields from each kidney section were photographed at 20× magnification. Image J (National Institutes of Health, USA) was used to quantitate the staining area percentage.