Technical Reference #1938

1938.

Polymorphisms in Human Organic Anion-transporting Polypeptide 1A2 (OATP1A2) Wooin Lee; Hartmut Glaeser; L. Harris Smith; Richard L. Roberts; Gilbert W. Moeckel; Guillermo Gervasini; Brenda F. Leake; Richard B. Kim, Vanderbilt University, Journal of Biological Chemistry, 280(1938), (2005)
Link To Paper

Materials & Methods:
Immunofluorescence Confocal Microscopy—HepG2 and HeLa cells were grown on sterile uncoated 35-mm glass-bottomed microwell dishes (MatTek Ashland MA) and transfected at 70–80% confluency with 2 g of V5-tagged wild-type or variant SLCO1A2 alleles (SLCO1A2*1–*7) using Lipofectamine 2000 (Invitrogen). After 48 h cells were fixed for 10 min in ice-cold 70% methanol. Cells were then permeabilized for 10 min in PBS containing 0.3% Triton X-100. After rinsing with PBS cells were placed in blocking buffer (2% bovine serum albumin in PBS) for 1 h at room temperature. Cells were then incubated with monoclonal antibody against V5 epitope (diluted 1:500 in blocking buffer) for 2 h at room temperature. After three washes in PBS containing 0.05% Tween 20 cells were incubated with secondary goat anti-mouse antibody labeled with the fluorescent dye Texas Red (Molecular Probes Eugene OR) for 30 min at 37 °C. During the final washes in PBS containing 0.05% Tween 20 SYTOX® Green (Molecular Probes Eugene OR) was added for nucleic acid staining. Confocal microscopy was performed with a Zeiss Axiovert 100-M inverted microscope equipped with a LSM510 laser scanning unit. A Zeiss 63 1.4 numerical aperture plan Apochromat oil immersion objective was used for all experiments. Confocal images were obtained using single excitation (595 nm) and emission (610–630 nm Texas Red) filter sets. Cells transfected with the parental plasmid lacking any insert were used as control. Cells transfected with V5-tagged SLCO1A2*1 plasmid without incubation with primary antibody were also used as an additional control. For slow frame scanning confocal images were obtained by scanning either laterally (top view x-y scans) or axially (side view x-z scans) across the cell. Image analysis and processing were performed with Zeiss LSM and Adobe Photoshop software.

Microscopic Technique
Immunofluorescence Confocal Microscopy

Cell Type(s)
HepG2 and HeLa cells