Biotinylated secondary anti rabbit igg antibodies

Immunohistochemistry (IHC) staining was performed as previously described^{28 (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 citric buffer, pH 6.0. 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 α-SMA (A2547, 1:20,000, Sigma-Aldrich, MIS, USA,) at 4 °C overnight. After washing with TBST for 5 min, the slides were incubated with biotinylated secondary anti-rabbit IgG antibodies (Dako) for 30 min. The slides were washed again with TBST before staining with HRP-conjugated streptavidin (Dako) for 10 min. Using a light microscope (Leica DM750 photomicroscope with ICC50W digital camera), six consecutive non-overlapping fields from each kidney section were photographed and the percentage of staining area was quantitated using the Image J software (National Institutes of Health, USA).

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.