Trichrome staining kit

The degree of renal fibrosis was determined by Trichrome staining kit (860–031; Ventana, Medical Systems, Inc. Roche Diagnostic USA). For the evaluation of renal injury score, ten tubulointerstitial fields were randomly selected and examined in terms of tubular dilatation, tubular atrophy, and interstitial fibrosis. Renal injury score was semi-quantitatively calculated based on the percentage of involved area with an assigned value: 0, none; 1, <10%; 2, 10% to 25%; 3, 25% to 75%; and 4, >75% as described elsewhere [24 (link)].
Immunohistochemistry (IHC, Ventana Medical Systems, Inc) was performed as described in our previous study [22 (link)]. For the specific method, we followed the manufacture’s instruction. Paraffin tissue sections (4μm) were analyzed for immunohistochemistry using monoclonal antibodies against α-SMA (ab5694; Abcam, Cambridge, MA, USA), E-cadherin (sc-7870; Santa Cruz Biotechnology, CA, USA), fibronectin (ab6328; Abcam Cambridge, MA, USA) and Type IV collagen (sc-9301; Santa Cruz Biotechnology CA, USA).
Ten individual high-power fields (magnification, 200 x) per kidney were analyzed, and representative images were presented.

Tissues were fixed in 15% buffered formalin for 1 week and decalcified for 5 days. After obtaining three 2 mm thick sections from the laminectomy area, the sections were embedded in paraffin. Sections of 5 μm were obtained axially and stained with Masson’s trichrome (Ventana, Trichrome Staining Kit, Tucson, Arizona, USA) using an automated slide stainer (Benchmark, Ventana Medical Systems Inc, Tuscon, AZ, USA). The slides were then examined using a microscope (Zeiss Axio Imager 2, Göttingen, Germany) and photographed with a camera (Zeiss AxioCam ERc 5s, Göttingen, Germany). Arachnoidal invasion and EF were blindly evaluated by a pathologist. The grading system of He et al. was used in the evaluation of EF [18]. Mean values ​​were obtained for the statistical evaluation (Table 1).