Pdgfrα

Adipose tissue samples were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and preincubated with quenching buffer (3% H₂O₂ in methyl alcohol) and blocking buffer (10% normal goat serum in PBS) for 30 min at room temperature. The samples were then treated with the primary antibodies against adiponectin (1:100, Thermo Scientific, IL, USA), PDGFRα (1:50, R&D System, MN, USA), UCP1 (1:300, Abcam, Cambridge, UK) and BrdU (1:100, Abcam) and with secondary Alexa Fluor 488- or 594-conjugated antibodies (Invitrogen). To detect BrdU staining, paraffin sections were incubated in 50% formamide 2× SSC (Welgene Inc., Daegu, Korea) for 2 h at 65 °C, washed with 2× SSC, and treated with 2 N HCl for 30 min at 37 °C. After washing with PBS, tissue sections were blocked with 10% normal goat serum (Vector Laboratories CA, USA). For nuclear staining, the samples were mounted on cover slips in DAPI-containing mounting medium (ImmunoBioScience, Burlingame, WA, USA). Images were captured using a confocal microscope system (LSM 710, Carl Zeiss, Oberkochen, Germany).

Histological and immunohistochemical analyses were performed on paraffin sections in which we observed the presence of RN by H&E staining. Each section was immunostained with the following antibodies: PDGF-A (1:20; R&D Systems, USA), PDGF-B (1:20; Abcam, Japan), PDGF-C (1:100; R&D Systems), PDGF-D (1:50; R&D Systems), PDGFR-α (1:20; R&D Systems), and PDGFR-β (1:50; R&D Systems) (Table 2). We routinely use a pressure cooker for 4 minutes to retrieve all the antigens. Endogenous peroxidase was blocked with 0.03% hydrogen peroxide for 40 minutes at room temperature. We used the ABC technique (Vector Laboratories, USA) for all of these antigens, before DAB (3, 3′ diaminobenzidine tetrahydrochloride (Wako Pure Chemical Industries, Japan)). The sections were counterstained with hematoxylin 3G (Sakura Finetek, Japan) and mounted.

C57BL/6 and Pdgfra^{tm11(EGFP)Sor}/J bladders were cut open from the neck up to the dome. Tissues were dissected in Krebs ringer solution containing 118.5 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl₂, 1.2 mM MgCl₂, 23.8 mM NaHCO₃, 1.2 mM KH₂PO4 and 11 mM dextrose, then pinned down on Sylgard dish and stretched 150% from the resting state. For urothelial denudation, urothelium was removed and surface of the muscle was scraped to remove any residual sub‐urothelial cells. Fixation and incubation of tissues were identical as previously described^{16 (link)}. For double labelling studies, tissues were re‐blocked for 1 h in 10% normal donkey serum (Sigma‐Aldrich) and incubated overnight in antibody of choice, diluted in 0.5% Triton‐X (Sigma‐Aldrich) and incubated in the appropriate Alexa Fluor (Invitrogen) antibody diluted 1:1000 in PBS for 1 h. Processed tissues were mounted with Aqua mount mounting media (Lerner Laboratories, Pittsburgh, PA, USA) on glass slides and cover slipped and imaged. The primary antibodies of PDGFRα (R&D Systems, Inc.) and SK3 (Alamone Labs) were used and primary antibodies were omitted in the procedure for negative controls.

The specimens were washed by cold PBS solution, the tissue cells were decomposed by cell lysate liquid, and protease inhibitor (Roche) and phosphatase inhibitor were added. The protein concentration was determined by the quinoline carboxylic acid method (BCA) (Pierce, Rockford, IL). The proteins were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to a PVDF membrane (Millipore, Billerica, MA). Sealed was done by TBS solution and 5% skimmed milk powder containing 0.1% Tween 20. Appropriate dilution of β-actin (Proteintech; HRP-60008, 1:5000), PDGFRα (R&D; AF1062, 1: 200), AKT1/2/3 (Santa Cruz; sc-8312, 1:1000), P-AKT (Thr308) (Cell signaling; 13038, 1:1000), cyclin D1 (Santa Cruz; sc-20044, 1:1000), C-myc (Santa Cruz; sc-40, 1:1000), MET (Santa Cruz; sc-8057, 1:1000), EGFR (Santa Cruz; sc-373746, 1:1000) of primary antibody in blocking buffer, and then the membrane was placed in a refrigerator overnight at 4°C. Removed the membrane and washed 3 times (10min per time). And added with the secondary anti-buffer (1: 5000, Pioneering Biotechnology, China), sealed at room temperature for 1h. At the end of the wash, the protein blots were detected by light emission (Millipore, Billerica, MA). Western blot grayscale values were quantified using Adobe Photoshop CS6 software.