Dm750 photomicroscope

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.

Tissues samples were fixed in 10% neutral formaldehyde, dehydrated in graded alcohol, embedded in paraffin wax, and then sectioned. After deparaffinizing and staining with periodic acid–Schiff (PAS) and hematoxylin and eosin (H&E), 4 µm-thick sections were examined under a Leica DM750 photomicroscope (Leica Microsystems, Heerbrugg, Switzerland). Up to ten randomly selected separate nonoverlapping microscopic fields for each kidney section were examined under light microscope at 400× magnification. H&E staining was used to evaluate endothelial proliferation, mesangial proliferation, matrix accumulation, and interstitial inflammation, and PAS staining was examined for interstitial fibrosis. The severity of injury was graded (3 rats/group) by an experienced pathologist who was unaware of the treatment groups using a modified method previously described [25 (link),26 (link)]. Arbitrary scores (au) were applied as follows: no change (0), no change or histopathological changes <10%; mild (0.5), 10–25%; moderate (1.0), 25–50%; and severe (1.5), >50%. Then, a mean score was calculated.