Eclipse fluorescence microscope

The induction of DNA damage was analysed using comet assay [21 (link),22 (link),23 (link)]. After treatment, the cells were resuspended in 0.75% low-melting-point agarose and spread onto microscope slides pre-coated with 0.5% normal-melting-point agarose. The slides were immediately put on ice, and the cells were lysed in ice-cold lysis buffer (2.5 M NaCl, 100 mM EDTA, 1% Triton X-100, 10 mM Tris, pH 10) for 1 h. The slides were incubated in an ice-cold developing solution (300 mM NaOH, 1 mM EDTA, pH > 13) for 20 min, followed by electrophoresis in ice-cold electrophoresis solution (30 mM NaOH, 1 mM EDTA, pH > 13) for 20 min at an electric field strength of 0.73 V/cm (32 mA). Then, the slides were washed with H₂O and stained with 4 µg/mL DAPI. The comets were observed at 200× magnification in an Eclipse fluorescence microscope (Nikon, Tokyo, Japan) attached to a COHU 4910 video camera (Cohu, San Diego, CA, USA) equipped with a UV-1 filter block (an excitation filter of 359 nm and a barrier filter of 461 nm) and connected to a personal computer-based image-analysis system Lucia-Comet version 4.51 (Laboratory Imaging, Prague, Czech Republic). One hundred comets were randomly selected from each sample, and the percentage of DNA in the tail (tail DNA (%)) was measured.

Gs22a cells in a six-well plate were transiently transfected with PTH1R-WT, PTH1R-R485X, PTH1R-E35K, PTH1R-Y132S, or PTH1R-H223R, each with an extracellular HA-tag, and 24 hours later were reseeded in a six-well plate onto glass coverslips, and processed for imaging at 48 h-post transfection. To visualize receptors on the surface of non-stimulated cells, cells were fixed with 3.7% paraformaldehyde for 5 min at room temperature, incubated for 1 hour at room temperature with anti-HA mouse monoclonal primary antibody (anti-HA.11, BioLegend, Cat. No. 901513), rinsed, incubated for 1 h at room temperature with a goat-anti-mouse IgG secondary antibody poly-conjugated to HRP (poly-HRP-anti-mIgG; ThermoFisher Scientific, Cat. No. 32230), stained with Tyramide-Alexafluor-594 (Tyr⁵⁹⁴; Tyramide SuperBoost Kit ThermoFisher Scientific, Cat. No. B4092), then mounted on a glass microscope slide in Vectashield containing DAPI and imaged using a Nikon Eclipse fluorescence microscope at 400X magnification.
To visualize receptors remaining on the cell surface after stimulation with ligand, cells were first stimulated with PTH(1-34)^FAM (30 nM) for 30 min at room temperature, then rinsed, fixed, and immuno-stained using anti-HA.11 primary antibody, poly-HRP-anti-mIgG secondary antibody and the Tyr⁵⁹⁴ SuperBoost reagents, as described above for surface receptors in non-stimulated cells.

Cardiomyocytes at 2 DIV were fixed with 4% paraformaldehyde for 15 min and then permeabilized with 0.25% Triton™ X-100 for 15 min under gentle shaking. After blocking with 5% goat serum in PBS for 30 min, cells were incubated with anti-cTnT (1:400) antibody overnight at 4 °C. Cells were then incubated with Alexa Fluor^® 488-conjugated goat anti-rabbit (1:500) secondary antibody for 2 h at RT. After aspirating secondary antibody, a concentration of 2 μg/mL of Hoechst 33342 was added to stain the nuclei. Images were taken using a Nikon eclipse fluorescence microscope using FITC filter and DAPI filter.