Lsm 710 meta confocal microscope

Cryosection of kidneys were fixed with 4% paraformaldehyde (Sigma), permeabilized and stained with primary antibodies against GSK3β (Cell Signaling), SYNPO (Santa Cruz), Podocin (Santa Cruz), WT1 (Santa Cruz), Desmin (Santa Cruz), followed by Alexa fluorophore-conjugated secondary antibody staining (Life Technologies). Finally, sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI), mounted with Vectashield mounting medium (Vector Laboratories), and visualized using a fluorescence microscope (BX43, Olympus, Tokyo, Japan) or a Zeiss LSM710 Meta confocal microscope (Carl Zeiss AG, Cologne, Germany). For dual-color staining, images were acquired sequentially to avoid dye interference. ImageJ software was used for post-processing of the images, e.g., scaling and merging.

Freshly isolated mouse stomach or pancreas was fixed in 4% (wt/vol) paraformaldehyde for 24 hours at 4°C and cryoprotected via sucrose gradient, starting with 15% sucrose for 6 hours at room temperature and finishing with 30% sucrose for 24 hours at 4°C. The tissue was then embedded in optimum cutting temperature compound before slicing. Tissue sections (7 μm) were blocked in 10% donkey serum, 0.05% Tween 80 for 1 hour at room temperature. Sections were then incubated with primary antibodies against SST (1:1000; Dako) and green fluorescent protein (GFP) (1:1000; Abcam) overnight at room temperature (for antibodies, see Table 1). Sections were rinsed with 5% donkey serum, 0.05% Tween 80 before being incubated for 1 hour at room temperature with Alexa Fluor 488 (1:300) and Alexa Fluor 555 (1:300) secondary antibodies (Invitrogen). Tissue stained with secondary antibodies only served as controls. Images were taken using a Zeiss LSM 710 META confocal microscope with ZEN 2010 software (Carl Zeiss) using a 63x/NA 1.4 objective at an optical slice thickness of 1.0 μm.

Live-cell imaging was performed with CHO-A₃ or CHO-A₃-GFP cells grown in Nunc Labtek 8-well plates and maintained as described above. For the ligand-binding experiments, the cells were incubated with the required concentration of fluorescent ligand for 10 min at 37°C before imaging. Binding specificity was assessed by preincubating cells with the nonfluorescent A₃AR antagonist MRS 1220 (100 nM) for 30 min at 37°C before the addition of CA200645. Images were captured with a Zeiss LSM710META confocal microscope (Zeiss, GmbH, Jena, Germany) fitted with a Plan-Apochromat ×63, 1.40 NA, DIC, oil-immersion objective (Zeiss). A 488 nm argon laser was used to excite the GFP, and a 633 nm HeNe laser was used to excite the BODIPY 630/650-labeled CA200645. A variable spectral detection system was used to capture emission at 480–530 and 645–680 nm for GFP and CA200645, respectively. Images within each set of experiments were collected by using identical settings for pinhole diameter, laser power, detector gain, and offset.