Picrosirius red solution

All paraffin embedded blocks were cut at 4 μm and the neighboring sections were used. The slides were dewaxed and rehydrated with xylene and ethanol. The samples were applied with adequate Picro Sirius Red solution (Sigma-Aldrich, USA) for 1 h or stained with Masson’s trichrome [18 (link)] or Verhoeff-Van Gieson elastic staining (EVG) [19 ] as previously described. Glass coverslips were applied using mounting medium. We photographed the whole mice tissue in the slides by microscopy (Nikon, USA). Each subject sample (n = 4 per group) was obtained 4–6 pictures of 40X magnification and analyzed. All pictures as RGB 8-bit resolution images were quantified by image-analysis system (Image Pro-Plus, Media Cybernetics, USA) [20 (link)]. The red color of Picro Sirius Red staining and the blue color of Masson’s trichrome staining were defined as fibrosis area. The image result was a measurement of the fibrosis area in the stained field being examined. The software system automatically calculated the mean fibrosis amount as a percentage (%).

Renal biopsy tissue underwent standard histopathological processing. In addition, tissue blocks were stained with Picro-Sirius Red Solution (Sigma-Aldrich, Direct Red 80) for Collagens I and III. Sirius red slides were uploaded to Slidepath and analysed offline using Tissue Studio 4.0 software (Definiens, Munich, Germany), a dedicated software package for quantitative digital pathology. A region-of-interest (ROI) of cortical tissue in each slide was drawn to exclude non-interstitial structures (medulla, capsule, fat, glomeruli and arterioles). Following this, the Tissue Studio 4.0 software automatically quantified the percentage of red staining (collagen) as a measure of cortical interstitial fibrosis (IF).

Tissue slides were stained as previously described on the paraffin‐embedded slides (Damhofer et al., 2015). Antibodies and dilutions used were CK19 1 : 500 (MU246‐UC; Biogenex), CXCR4 1 : 400 (Ab124824; Abcam, Cambridge, UK), Ki67 1 : 2000 (SAB5500134; Sigma). For picrosirius red staining, slides were deparaffinized, stained in a 0.1% picrosirius red solution (Sigma) in saturated picric acid for 1 h, and washed three times with 0.1 m acetic acid solution. All slides were imaged on an Olympus BX51 (Tokyo, Japan). Quantification of Ki67 staining was performed with Fiji count particles (Schindelin et al., 2012), after DAB/H color deconvolution. For IF images, slides were cut at 10 μm, mounted in Prolong Gold (ThermoFisher), and imaged on an EVOS fluorescence microscope (ThermoFisher). For collagen staining in tissue culture vessels, cells were cultured as described and after 7 days of coculture washed with PBS three times prior to fixation in 4% paraformaldehyde for 15 min. Following three washes with PBS, cells were stained for their collagen deposition as described above for 18 h and were equally treated as the tissue slides. Cells were subsequently imaged on an Olympus BX51. Quantifications of the percentage of Ki67‐positive nuclei, width of HE staining, or percentage of Venus‐positive cells were performed using Fiji package of imagej.