Csu x1 spinning disc confocal microscope

Tg(myl7:GCaMP)^s878 adult zebrafish were outcrossed to wild-type strain and fertilized eggs at 1-cell stage were injected with 2 ng of the morpholino oligomer tnnt2a (5′-CATGTTTGCTCTGATCTGACACGCA-3′). Larvae at 24 h post-fertilization were placed in 0.003% N-phenylthiourea to prevent pigmentation. For nifedipine titration, 3 dpf larvae were incubated in E3 or E3_0Ca medium containing 10, 25, or 100 µM nifedipine for 30 min. Then, larvae were embedded in 1% low melting point agarose and transferred to an 8-well glass bottom plate (ibidi, Gräfeling, Germany). To study the recovery of Ca²⁺ dynamics with GCaMP, the aequorin reconstitution protocol described above was applied to 3 dpf larvae, without addition of CTZ. Fluorescence images were acquired at a rate of 200 Hz with a CSU X1 spinning disc confocal microscope (Carl Zeiss, Oberkochen, Germany) equipped with a Hamamatsu ORCA Flash4.0 sCMOS camera (Hamamatsu Photonics, Japan) in 16 bits with 2×2 binning.

Tg(myl7:GCaMP)^s878 adult zebrafish were outcrossed to wild-type strain and fertilized eggs at 1-cell stage were injected with 2 ng of the morpholino oligomer tnnt2a (5'-CATGTTTGCTCTGATCTGACACGCA-3'). Embryos from 24 hours post-fertilization (hpf) were maintained in 0.003% N-phenylthiourea to prevent pigmentation. Larvae were embedded in 1% low melting point agarose and transferred to an 8-well glass bottom plate (ibidi, Germany). Fluorescence images were acquired at a rate of 200 Hz with a CSU X1 spinning disc confocal microscope (Carl Zeiss, Germany) equipped with a Hamamatsu ORCA Flash4.0 sCMOS camera (Hamamatsu Photonics, Japan) in 16 bits with 2 x 2 binning. For image analysis, ROIs were drawn in the atrium and in the ventricle to obtain mean intensity values. An exponentially weighted moving average smoothing with a smoothing factor of 0.7 was applied and data was transformed into ΔF/F₀ = (F_t - F₀)/F₀; where F_t is the fluorescence at a given time and F₀ is the minimum diastolic fluorescence value. For characterization of the Ca²⁺ transients, ΔF/F₀ data were analyzed with Clampfit 10.7 (Molecular Devices, CA, USA) to determine rise time 10% to 90% and decay time 90% to 10%.