(micropuncture) and (proximal tubule cell line) research showed that transcytosis. (LTA).

(micropuncture) and (proximal tubule cell line) research showed that transcytosis. (LTA). Opossum Kidney (Okay) Cells Okay cells were cultured and managed in high-glucose (450 mg/dl) Dulbeccos revised Eagles medium (DMEM) supplemented with 10% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 g/ml) as explained.21 For transcytosis study, OK cells were seeded on Transwell, and grown to complete confluence for 2 days (typical transepithelial resistance approximately 1C5 k).72 For uptake study, fluorescein-labeled mice and mice were sacrificed 24 hours after injection. Kidneys were harvested and fixed with 2.5% paraformaldehyde via aortic perfusion, eliminated, and post-fixed in 4% paraformaldehyde (4C for 4 hours). Immunogold labeling of ultrathin freezing tissue sections was performed as explained.21 Kidney cortex was infiltrated with 2.3 M sucrose overnight, frozen in liquid nitrogen, and 70C80-nm-thick sections were made (Ultramicrotome Reichert Ultracut E; Leica Microsystems, Wetzlar, Germany) and mounted on Formvar-coated nickel grids. The sections were incubated with KM2076 antibody and followed by incubation with gold-conjugated protein A (10-nm gold particles, Sigma-Aldrich) for 60 moments. After staining with uranyl acetate, sections were visualized with Jeol 1200 Ex lover transmission electron microscope (Jeol Ltd., Akishima, Japan). Statistical Analyses Data are indicated as the meansSD. Statistical analysis was performed using unpaired test, or paired test, or ANOVA followed by StudentCNewmanCKeuls test whenever appropriate. A P-value of0.05 was considered statistically significant. Unless Gpc4 otherwise stated, representative numbers reflect the results in a minimum of three self-employed experiments. Disclosures None. Supplementary Material Supplemental Data: Click here to view. Acknowledgments This work was in part supported from the National Institutes of Health (R01-DK092461, R01-091392, and R01-DK13686), OBrien Kidney Study TKI-258 Center in the University or college of Texas Southwestern Medical Center TKI-258 (P30-DK07938), American Heart Foundation Western Affiliate Beginning-Grant-in-Aid (0865235F), the TKI-258 Simmons Family Foundation, the Charles and Jane Pak Basis, the Pak Center Innovative Study Support, and the Canadian Institutes of Health Study (MOP-93725). The authors would like to say thanks to Dr. Philipp Scherer for assistance with infrared dye labeling and for providing infrared scan system, and Drs. Michel Baum, Jyothsna Gattineni and Xiao Yan for helpful discussions during the preparation of the manuscript. Authors are thankful to Ms. Jean Paek for technical support, and to Ms. Carolyn Griffith for assistance in enrollment of study subjects. A.B. was in part supported by Visiting Scholar Honor from National Natural Science Basis of China (81170660H0509, 81270408H0220), and Provincial Organic Science Basis of Jiangsu, China (BK2011849). Footnotes Published online ahead of print. Publication day available at www.jasn.org. This short article contains supplemental material on-line at TKI-258 http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2014101030/-/DCSupplemental..