Ab70287

Renal histology was assessed by staining 5 µm-thick kidney sections with Masson’s Trichrome stain and analysed through microscopy. Interstitial fibrosis was quantified by measuring collagenous fibrotic areas stained in 10 random cortical fields/section from images taken at a magnification of 40× using multiphase image analysis with ImageJ software version 1.53c [55 (link)].
Immunofluorescence staining was performed as follows. Histological sections (5 μm thick) were deparaffinised, hydrated, and subjected to antigen-retrieval. The slides were blocked with 3% BSA/PBS for 30 min, permeabilised in 0.2%Triton-X100/PBS for 6 min at 4 °C, and then incubated with the primary antibodies: anti-non phospho active β-catenin (1:100, 8814s, Cell Signaling Technology, Danvers, MA, USA), anti-NGAL (1:100, ab70287, Abcam, Cambridge, UK), and anti-CD45 (1:100, ab10558, Abcam, Cambridge, UK) overnight at 4 °C. After three washes with PBS, the sections were incubated with Alexa Fluor 488 fluorescence secondary antibody (1:1000, Life technology, New York, NY, USA) and Alexa Fluor Texas red (1:1000, Life technology, New York, NY, USA) for 1 h at room temperature. Following three washes, the sections were stained with DAPI, mounted with fluorescent mounting media, and analysed via microscopy. Sections labelled with only secondary antibodies served as controls

Kidney tissues were fixed in 10% formalin, dehydrated, and embedded in paraffin for hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS). The degree of tubular injury was scored based on the percentage of injured tubules: 0, 0%; 1, ≤10%; 2, 11–25%; 3, 26–45%; 4, 46–75%; and 5, 76–100% [20 (link),21 (link)]. IHC staining was conducted using antibodies against neutrophil gelatinase-associated lipocalin (NGAL; 1:100; ab70287; Abcam, Cambridge, MA, USA), 4-hydroxynonenal (4-HNE; 1:100; ab48506; Abcam, Cambridge, MA, USA) or galectin-3 (1:100; ab2785; Abcam, Cambridge, MA, USA). Then, the sections reacted with HRP-conjugated secondary antibodies. The FITC-labeled lotus tetragonolobus lectin (LTL; 1:100; FL-1321; Vector Laboratories, Burlingame, CA, USA) was used to detect the brush border of proximal tubules. Nuclei were counterstained with DAPI. Quantification of positive staining for NGAL, 4-HNE, or LTL was analyzed using an image-analyzing software (IMT i-Solution, Coquitlam, BC, Canada) in 10 random fields per sample. The number of galectin-3-positive cells was counted in 10 random fields per sample.