Abc reagent

Human kidney samples were collected from surgical nephrectomies performed at Helsinki and Uusimaa Hospital district, and represented the non-malignant part of the kidney. Samples were fixed with 10% formalin, dehydrated, and embedded in paraffin. Immunoperoxidase staining was performed with a VectaStain Elite kit (Vector Laboratories, Burlingame, CA, USA). Sections were deparaffinized, antigen retrieval was performed by boiling in a microwave oven in 10 mM citric acid, pH 6.0 for 15 min, and endogenous peroxidase was inactivated by incubation in hydrogen peroxide in methanol for 30 min. Sections were blocked with CAS-block (Invitrogen) and incubated with primary antibodies diluted in ChemMate™ (DakoCytomation, Glostrup, Denmark) and with biotinylated secondary antibodies followed by incubation with ABC-reagent and AEC (Sigma-Aldrich) for colour development. Sections were counterstained with hematoxylin, mounted with Shandon Immu-Mount (Thermo Scientific, Waltham, MA, USA) and photographed using Nikon Eclipse 800 microscope. The use of human material was approved by the local Ethics Committee.