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 E30Ca 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.
8 well glass bottom plate
Manufactured by Ibidi
Sourced in Germany
The 8-well glass bottom plate is a laboratory equipment designed for cell culture applications. It features a glass bottom that provides optical clarity for high-resolution imaging of cells. The plate has 8 individual wells, allowing for multiple samples to be cultured and observed simultaneously.
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5 protocols using 8 well glass bottom plate
1
Nifedipine-Induced Calcium Dynamics in Zebrafish
2
Calcium Imaging in Zebrafish Heart
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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 = (Ft - F0)/F0; where Ft 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%.
3
Nanoparticle Diffusion in Simulated Vitreous
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NP diffusion through the TSH was evaluated by confocal imaging using the optimized SV as a model. The SV was heated to room temperature, then 200 µL was put in the wells of an 8-well glass-bottom plate (Ibidi, Gräfelfing, Germany), covered with Parafilm, and incubated at 37 °C for 1 h. After that, 20 µL of cold (4 °C) Cy5-labeled NP suspension or NP-loaded TSH was injected into the SV. Confocal imaging started right after the injection, keeping the temperature at 37 °C. The laser was set to acquire the signal of the Cy5 (ex. 650 nm; em. 670 nm), and images were acquired every 6 h for a total of 48 h using a 10x objective. Each time point consisted of 35 Z-stack images with a 5 µm Z-step. Two fields were imaged, i.e., the injection site and the opposite corner of the well (5 mm distance). After acquisition, images were processed with Fiji ImageJ: Z-stacks were collapsed to maximum intensity per each time point. A threshold was set to remove background signal, and total fluorescent intensity was measured for each time point and for each field. Each sample was analyzed in triplicate. Fluorescence intensity% was calculated as follows:
4
Aequorin-based Cardiac Bioluminescence Imaging
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After aequorin reconstitution, Tg(cmlc2:GA) embryos were embedded in 100 µL of 0.3% low melting agarose prepared in E3 medium and transferred to an 8-well glass bottom plate (ibidi, Gräfelfing, Germany). When the agarose solidified, 100 µL of E3 medium was added and a small portion of agarose surrounding the heart of the embryos was cut out to improve diffusion of drugs and Triton X-100. For the signal-to-noise ratio (SNR) calculation, bioluminescence images were acquired at 9 Hz for 1 min. For longer recordings, drugs were added after 1-10 min basal tracking.
At the end of every recording Triton X-100 (5%) was added to release all luminescence counts from the remaining active aequorin. Bioluminescence images were acquired as previously described [1] (link) with a custom-built microscope housed in a light-tight box to maintain complete darkness during imaging. Images were acquired continuously in 16 bits with 4 x 4 binning, 255
EM gain, at a rate of 25, 17, 12, 9, 2 or 1 Hz (frames/s) with an EM-CCD camera (Hamamatsu Photonics, Hamamatsu, Japan). The spatial resolution of the images was 12.8 µm x 12.8 µm/pixel and the total field of view was 1638 µm x 1638 µm.
At the end of every recording Triton X-100 (5%) was added to release all luminescence counts from the remaining active aequorin. Bioluminescence images were acquired as previously described [1] (link) with a custom-built microscope housed in a light-tight box to maintain complete darkness during imaging. Images were acquired continuously in 16 bits with 4 x 4 binning, 255
EM gain, at a rate of 25, 17, 12, 9, 2 or 1 Hz (frames/s) with an EM-CCD camera (Hamamatsu Photonics, Hamamatsu, Japan). The spatial resolution of the images was 12.8 µm x 12.8 µm/pixel and the total field of view was 1638 µm x 1638 µm.
5
Visualizing Calcium Dynamics in Zebrafish
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GCaMP fluorescence imaging was carried out at the Max Planck Institute for Heart and Lung Research (Bad Nauheim, Germany). Tg(cmlc2: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 at 24 hours postfertilization were placed in 0.003% N-phenylthiourea to prevent pigmentation. For nifedipine titration, 3 dpf embryos were incubated in E3 medium or zero Ca 2+ E3 medium containing 10, 25 or 100 µM nifedipine for 30 min. Then, embryos were embedded in 1% low melting point agarose and transferred to an 8-well glass bottom plate (ibidi). To study the recovery of Ca 2+ dynamics with GCaMP, the aequorin reconstitution protocol described above without CTZ was applied to 3 dpf embryos. Fluorescent 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 x 2 binning.
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