Hpa002083

Original paraffin blocks of foetal and adult kidneys, precursor lesions as well as a TMA containing PRCCs were used for this study. TMA was constructed by one of the authors (GK) as described earlier 11 (link). The 4µm sections placed onto FLEX IHC microscope slides (DAKO, Glostrup, Denmark), dewaxed in xylene and rehydrated in graded ethanol. Antigen retrieval was performed by boiling the slides in 10 µM sodium citrate buffer, pH 6.0 in 2100-Retriever (Pick-Cell Laboratories, Amsterdam, The Netherlands). Endogenous peroxidase activity and nonspecific staining were blocked by incubation with 3% hydrogen peroxide containing 1% normal horse serum for 10min at room temperature.
Slides were then incubated overnight at 4°C in moist chamber with the rabbit polyclonal anti-MET antibody (sc-12, Santa Cruz Biotechnology, Inc.) and rabbit polyclonal anti-HNF1B antibody (HPA-002083, Sigma Aldrich, Inc.), both diluted 1:100. HRP conjugated anti-rabbit secondary antibody (MACH4 Universal HRP-Polymer, Biocare Medical, Concord, CA, USA) was applied for 30min and color was developed using the AEC or DAB substrates (DAKO). Tissue sections were counterstained with Mayer's hematoxylin and cover-slipped with Faramount (DAKO) or Pertex (Medite GmbH, Burgdorf, Germany). In negative controls, the primary antibody was omitted.