Aperio cso system

3μm sections from kidneys fixed in paraformaldehyde stained. For antigen retrieval, sections were either incubated with 0.1% trypsin (Sigma Aldrich), 0.8% collagenase or microwaved in citrate buffer (pH 6.0). Primary antibodies were incubated overnight at 4°C by adding rabbit anti-mouse collagen IV antibody (1:400, Millipore, Darmstadt, Germany), rat anti-mouse CD31 (1:50, Dianova GmbH, Hamburg, Germany), rabbit anti-mouse active β-catenin (1:600, Cell signaling, Danvers, MA, USA), goat anti-mouse nephrin (1:500, R&D), rabbit anti-mouse WT-1 (1:200, Novus), rabbit anti-mouse collagen I (1:500 dilution, Abcam), rabbit anti-mouse CD45 (1:400, Abcam) and rat anti-mouse F4/80 (1:600, Biorad, Hercules, CA, USA). Slides treated with nonspecific antisera instead of primary antibody were used as negative control.
Collagen I, CD31, active β-catenin, F4/80, nephrin and collagen IV stained slides were scanned using the Aperio CSO system (Leica Biosystems, Buffalo Grove, IL, USA). The positive area was analyzed using Aperio Imagescope software. WT1-positive nuclei per glomerulus and CD45 positive cells per high power field were counted.

Sections were deparaffinized and hydrated. For WT1 and ED1, sections were microwaved in citrate buffer (pH 6.0) for 5 min. Endogenous peroxidase was quenched with 3% hydrogen peroxidase for 10 min, and slides were then exposed to Power Block (BioGenex Laboratories, San Ramon, CA, USA) for 45 min. The primary antibodies used were rabbit anti-rat WT1 (1:800; Santa Cruz), rabbit anti-rat PAI-1 (20 μg/ml; American Diagnostica, CT, USA), mouse anti-rat ED1 (1:50; Dako) and mouse anti-rat collagen I (1:400; Abcam, Cambridge, MA, USA). Sections were incubated overnight at 4 °C. Immunoperoxidase staining was performed with the Vectastain ABC kit (Vector Laboratories, Burlingame, CA, USA) with diaminobenzidine as a chromogen. Hematoxylin was used as a counterstain.
WT1 and ED1-positive nuclei per glomerulus were counted, and localization was assessed based on cell anatomical location and morphology. Collagen I stained slides were scanned by using the Aperio CSO system (Leica Biosystems, Buffalo Grove, IL, USA). The positive area was analyzed by using Aperio Imagescope software. All sections were examined without knowledge of the treatment protocol.