Urat1

Immunohistochemistry study was performed using the streptavidin-biotin complex method (TAHC02D kit, Biotna, Kaohsiung, Taiwan). Paraffin-embedded fixed tissue sections (4 μm) were deparaffinized with xylene, and dehydrated with ethanol. Antigen retrieval was carried out with 10 mM citrate buffer (pH 6.0) twice for 5 min microwave treatment. The sections were incubated with 3% H 2 O 2 methanol for 10 min, and then incubated with 10% serum for 30 min at room temperature. Then, anti-SGLT1 (1:50, Abcam, Cambridge, UK), SGLT2 (1:50, Biorbyt, Cambridge, UK), GLUT 9 (1:100, Merck Millipore, Billerica, MA, USA), UAT (1:25, Abbiotec, San Diego, CA, USA), URAT1(1:100, Abbiotec, San Diego, CA, USA) antibody was added, and incubated at 4°C overnight. The sections were incubated with the secondary antibody at room temperature for 30 min, and then with peroxidase-conjugated streptavidin at 37°C for 30 min. The expression of proteins was visualized using 3, 3'-diaminobenzidine tetrahydrochloride at a concentration of 30 mg/mL, containing 0.03% H 2 O 2 . All sections were photographed under light microscopy (x400) with digital camera (DN100, E-600, Nikon; Tokyo, Japan). Immunostaining-positive areas were determined using Adobe Photoshop, and quantified in 20 random fields per section using NIH Image 1.62.