Horseradish peroxidase hrp conjugated streptavidin

Kidneys were fixed in 10% formalin for 36-h and embedded in paraffin. Paraffin sections were prepared at 4 μm thickness and mount on microscope slides. The sections were stained with hematoxylin and eosin (H&E) for general renal structure and perirenal fat visualisation. Immunohistochemistry (IHC) staining was performed as previously described^{55 (link)}. Briefly, the tissue sections underwent dewaxing (with xylene), rehydration (with ethanol), antigen-retrieval (99 °C for 20 min in 0.01M, pH 6.0 citric buffer), washing (with water), endogenous peroxidase deactivation with 3% H₂O₂ (Sigma-Aldrich, Dublin, Ireland), blocking (with Protein Block Serum-Free, Dako, Glostrup, Denmark), incubation with primary antibodies against fibronectin (1:1000, Abcam, Cambridge, UK) and collagen IV (1:1000, Abcam, Cambridge, UK) followed by biotinylated secondary anti-rabbit IgG antibodies and finally horseradish peroxidase (HRP)-conjugated streptavidin (Dako, Glostrup, Denmark). The tissue specimens were examined by brightfield microscopy (Olympus, Japan). Six consecutive non-overlapping fields from each section of renal cortex were photographed under high magnification. Image J (National Institutes of Health, USA) was used for estimation of the specific staining area. IHC score was determined by log transformation of the staining area.

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.