Journal: bioRxiv

Article Title: Tandem Requirement for Full Renal D 1 R and D 5 R Activity

doi: 10.1101/736611

Figure Lengend Snippet: A & B : HEK-293 cells were transiently transfected with D 1 R::RFP and D 5 R::GFP and then grown on a cover slip or in a polarized state in a Transwell ™ insert; the colocalization of these receptors was evaluated via laser scanning confocal microscopy. Wheat germ agglutinin (WGA) tagged with Alexa Fluor□ 647 was used to visualized the plasma membrane, while DAPI (#H-1500, Vector Laboratories, Burlingame, CA) was used to visualized the nuclei. Colocalization was observed as discrete areas of white (red+green+blue) or yellow (red+green). Serial images along the X, Y, and Z axes were obtained in cells grown in Transwell ® and stitched together to show 3D colocalization of the receptors. 630x magnification, scale bar = 10 μm, n=3 independent experiments. C : hRPTCs were double-transfected with D 1 R and D 5 R tagged with the C and N termini of the fluorescent protein EYFP, respectively. The cells were grown on cover slips for 48 hours post-transfection, serum-starved for 2 hrs, and then prepared for confocal microscopy. The interaction of the tagged receptors results in the reconstitution and fluorescence of EYFP (pseudocolored green). An overlay of the BiFC signal and the nucleus (pseudocolored blue) is shown to indicate the distribution of the D 1 R-D 5 R complexes, 630x magnification, scale bar =10 μm, n=3 independent experiments. Transfection of D 1 R-EYFP alone was used as negative control.

Article Snippet: The tissues were counterstained with wheat germ agglutinin (WGA) tagged with Alexa Fluor ™ 647 (#A-31573, ThermoFisher Scientific, Waltham, MA) and DAPI (#H-1500, Vector Laboratories, Burlingame, CA) to visualize the plasma membrane and nuclei, respectively.

Techniques: Transfection, Confocal Microscopy, Plasmid Preparation, Fluorescence, Negative Control

Journal: bioRxiv

Article Title: Tandem Requirement for Full Renal D 1 R and D 5 R Activity

doi: 10.1101/736611

Figure Lengend Snippet: A section of the human kidney was double immunostained for endogenous D 1 R (red) and D 5 R (green) ( A ). WGA (magenta) was used to visualize the plasma membrane, e.g., apical brush border of proximal tubules, while DAPI was used to visualize the nuclei. Colocalization in yellow is indicated by the arrows. DIC = differential interference microscopy. Sections of a mouse kidney infused with either vehicle (Basal) or fenoldopam was double immunostained for endogenous D 1 R (green) and D 5 R (red) ( B ). The RPT marker CD15 and DAPI were used to visualize the brush border and nuclei, respectively. Colocalization is indicated by the yellow or white areas in merged images. 630x magnification, scale bar=10 μm, n=3 independent experiments.

Article Snippet: The tissues were counterstained with wheat germ agglutinin (WGA) tagged with Alexa Fluor ™ 647 (#A-31573, ThermoFisher Scientific, Waltham, MA) and DAPI (#H-1500, Vector Laboratories, Burlingame, CA) to visualize the plasma membrane and nuclei, respectively.

Techniques: Microscopy, Marker