Imagescope v12

Renal sections underwent deparaffination and heat-mediated antigen retrieval (citrate buffer, pH = 6.00) as previously described (18 (link)). For caspase3 and Ki67 detection, sections were permeabilized with Triton 0.25% for 5 min, then blocked by Protein Block Solution (DakoCytomation, USA) for 10 min. Incubation was performed with antibodies against: Caspase-3 (Novus Biologicals, Abingdon Science Park, UK), PDGFRβ (Abcam, Cambridge UK), Ki-67 (Novus Biologicals), and detected by the Peroxidase/DAB Dako Real EnVision Detection System (Dako, Glostrup, Denmark). The peroxidase reaction was shown by a brown precipitate, counterstained with Mayers hematoxylin (blue). Negative controls were prepared by incubation with a control irrelevant antibody. Images were scanned by Aperio ScanScope CS2 device and signals were analyzed with the ImageScope V12.1.0.5029 (Aperio Technologies, Vista, CA, USA).

The cells were blocked for 1 h (BSA in PBS, pH 7.4) and then incubated with primary antibodies overnight at 4 °C or for 2 h at room temperature. The immune complexes were identified with the respective specific secondary antibodies for 1 h at room temperature. For caspase-3, cells were permeabilized with Triton 0.25% for 5 min, then blocked with protein block solution (Dako Cytomation, Santa Clara, CA, USA) for 10 min. Incubation was performed with antibodies against: caspase-3 (Novus Biologicals, Abingdon Science Park, Oxford, UK) and detected using the Peroxidase/DAB Dako Real EnVision Detection System (Dako, Glostrup, Denmark). The peroxidase reaction was shown by a brown precipitate counterstained with Mayers hematoxylin (blue). Negative controls were prepared via incubation with a control-irrelevant antibody. Images were scanned using an Aperio ScanScope CS2 device, and signals were analyzed with the ImageScope V12.1.0.5029 (Aperio Technologies, Vista, CA, USA).

Renal sections underwent deparaffination and heat-mediated antigen retrieval (citrate buffer, pH=6.00) as previously described. For p16, p21 and βcatenin detection, sections were permeabilized with Triton 0.25% for 5 min, then blocked by Protein Block Solution (DakoCytomation, USA) for 10 min. Incubation was performed with antibodies against: p16, p21 (Abcam, Cambridge UK), IL-6 (Novus Biologicals), wnt4 and βcatenin. Then, the positive staining was detected by the Peroxidase/DAB Dako Real EnVision Detection System (Dako, Glostrup, Denmark). The peroxidase reaction was shown by a brown precipitate, counterstained with Mayers hematoxylin (blue). Negative controls were prepared by incubation with a control irrelevant antibody. Images were scanned by Aperio ScanScope CS2 device and signals were analyzed with the ImageScope V12.1.0.5029 (Aperio Technologies, Vista, CA).