Sections were pretreated in the same way as described above. HIF-1α was then immunostained using the streptavidin-peroxidase technique. Briefly, after deparaffinization, antigen retrieval, and peroxidase quenching, slides were incubated with the primary antibody monoclonal anti-HIF-1α (H1alpha67; 1:200; Novus Biologicals, Littleton, CO, USA) at 4 °C overnight. Antibody binding was detected using an EliVision plus kit (EliVision™ superKIT-9922, Maixin, Fuzhou, China). Diaminobenzidine was used for staining, followed by a hematoxylin counterstain.
The HIF-1α immunohistochemical expression in the renal tubular epithelial cells was analyzed by a optical microscopy (× 400) and was scored using 0–4 points: 0 points, no or very weak nuclear staining; 1 point, < 25% of nuclear staining; 2 points, 25–50% of nuclear staining; 3 points, 50–75% of nuclear staining; 4 points, > 75% of nuclear staining [10 (link), 11 (link)].
The HIF-1α immunohistochemical expression in the renal tubular epithelial cells was analyzed by a optical microscopy (× 400) and was scored using 0–4 points: 0 points, no or very weak nuclear staining; 1 point, < 25% of nuclear staining; 2 points, 25–50% of nuclear staining; 3 points, 50–75% of nuclear staining; 4 points, > 75% of nuclear staining [10 (link), 11 (link)].
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