Qwin standard image analysis software

Serial sections 4 μm thick of frozen kidneys were fixed with 4% paraformaldheide for 8 minutes at +4°C. After rinsing in PBS and incubation with BSS for 60 minutes at RT, the sections were sequentially incubated with primary antibodies to detect E‐cadherin (Abcam, Cambridge, UK: green) and αSMA (Abcam, Cambridge, UK: red), or E‐cadherin (green) and S100A4 (red), or laminin (green) and αSMA (red) and then with the secondary antibodies conjugated with Alexa Fluor 488 and Alexa Fluor 594 for 24 hours. Subsequent exposure to DAPI allowed detection of the nuclei. The omission of the primary antibody confirmed the specificity of the immunoreaction. The analysis was performed by a single examiner blind to treatment, using QWin Standard Image Analysis software (Leica Microsystems GmbH, Wetzlar, Germany).

This analysis was performed by a single examiner who was kept blind to treatment, using a Leica DM photomicroscope equipped with QWin Standard Image Analysis software (Leica Microsystems GmbH, Wetzlar, Germany). Routines were specifically created to estimate immunoreactivity for E‐cadherin, αSMA, or S100A4 in an operator‐independent fashion. The entire cortical region was examined at magnification of ×20 in 6 sections for each rat, with about 300 views for each treatment. Vessels and glomeruli were excluded from the analysis. Immunoreactivity was quantified in each field as the percentage of total surface area. All sections were stained during the same experiment to minimize experimental variability and examined by a single operator blinded to treatments. The intra‐assay and interassay coefficients of variation were less than 5% and 10%, respectively.