Histological and Immunostaining Analysis of Kidney Tissue

The periodic acid-Schiff (PAS) staining was performed using the PAS staining kit (#395B-1KT; Sigma-Aldrich, St. Louis, MO, USA). For immunohistochemistry, human kidney sections were stained with the primary antibodies against DKK1 (sc-25516; Santa Cruz, Dallas, TX, USA), fibronectin (ab45688; Abcam, Cambridge, UK) or TGF-β1 (BS1361; Bioworld Technology, Dublin, OH, USA), whereas rat kidney sections were stained with antibodies against DKK1 (sc-25516; Santa Cruz, Dallas, TX, USA), TGF-β1 (E11264; Spring Bioscience, Pleasanton, CA, USA), collagen IV (BSB5355; BioSB, Santa Barbara, CA, USA), fibronectin (BS1644; Bioworld Technology, Dublin, OH, USA) or IL-1β (sc-7884; Santa Cruz, Dallas, TX, USA). For quantification of PAS and immunohistochemical staining, six areas from each section were analyzed using the Image-Pro Plus software (Media Cybernetics, Silver Spring, MD, Version 6.3). Immunofluorescence staining of rat kidney sections with the β-catenin antibody (GTX101435; GeneTex, Irvine, CA, USA) was conducted according to the previously described procedure [49 (link)]. The mean fluorescence intensity of β-catenin in kidney sections was quantified using the CellSens software package Version 2.1 (Olympus Medical Systems, Tokyo, Japan).