Leica bond system

Sections from paraffin embedded kidney biopsies with chronic kidney disease (normal and reduced eGFR) and kidney tissue obtained from tumor nephrectomy specimens (n = 54) were stained with antibodies against HIF1α, VEGFA, and ABCG2. After antigen retrieval, slides were incubated with the following antibodies: HIF1α clone mgc3 ab16066 (Abcam, Cambridge, UK), dilution 1:400; VEGFA sc-152 (Santa Cruz Biotechnology), dilution 1:300; ABCG2 ab3380 (Abcam, Cambridge, UK), dilution 1:60. The immunohistochemical staining was conducted on automated staining systems (ABCG2 on Ventana BenchMark, Ventana Medical Systems, USA; HIF1α and VEGFA on Leica Bond System, Leica Biosystems) following the manufacturer’s instructions. Visualization was performed using the avidin–biotin complex method leading to a brown staining signal. For all stainings the intensity in the glomerular and tubulointerstitial compartment (negative = score 0, weak = score 1, strong = score 2) in comparison to expression in normal tissue was assessed. Statistical analysis using Fisher’s exact test was performed with SPSS statistics software version 22 (IBM, USA). p-values < 0.05 were considered as significant.

Formalin-fixed, paraffin-embedded tumour sections (2.5 μm) were transferred to glass slides. Immunohistochemistry was carried out using the automated Leica BOND system and the Bond Polymer Refine Detection Kit (Leica Biosystems). G^o was visualized using a mouse monoclonal 8-oxoguanine primary antibody (ab64548, Abcam, diluted 1:250). The immunostained slides were scanned using the NanoZoomer Digital Slide Scanner (Hamamatsu Photonics).