Hoechst 33342, TMRM, calcein-AM, and Ethidium Homodimer-1 were all purchased from Biotium and applied using manufacturer’s protocol31 (link),57 (link). MitoSOX was purchased from Life Technologies and was applied to cells for a 10-min incubation to assess mitochondrial superoxide31 (link). Calcein-Cobalt Chloride (CoCl2) staining was used to assess mitochondrial permeability transition, which is achieved through quenching cytosolic calcein-AM signal with 5 μM CoCl2 described previously31 (link). Immunofluorescence with Anti-MF-20 (DSHB # AB_2147781) was used with fluorescent secondary antibody conjugated to Alexa Fluor 555 (Themo # A-31570) to assess cardiac hypertrophy in fixed and permeabilized PVNM cells. All imaging experiments were done on an Olympus IX70 inverted microscope with QImaging Retiga SRV Fast 1394 camera using NIS Elements AR 3.0 software. Quantification, scale bars, and processing including background subtraction, was performed on Fiji (ImageJ) software.
Retiga srv fast 1394 camera
Manufactured by National Instruments
The Retiga SRV Fast 1394 camera is a high-speed, high-resolution digital camera designed for scientific and industrial applications. It features a CCD image sensor and supports the IEEE 1394 (FireWire) interface for fast data transfer. The camera is capable of capturing images at a maximum resolution of 1360 x 1024 pixels and a maximum frame rate of 30 frames per second.
Automatically generated - may contain errors
2 protocols using retiga srv fast 1394 camera
1
Multiparametric Imaging of Cellular Stress Responses
2
Multimodal Imaging of Calcium Signaling
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TMRM, Calcein-AM, ethidium homodimer-1, MitoView Green, and Hoechst 33342 were purchased from Biotium. MitoSox was purchased from Life Technologies. MPTP imaging was performed by quenching the cytosolic Calcein-AM signal with 5 µM cobalt chloride during the incubation period. All imaging, including ER and mitochondrial calcium imaging, was done on an Olympus IX70 inverted microscope with QImaging Retiga SRV Fast 1394 camera using NIS Elements AR 3.0 software. Quantification, scale bars, and processing including background subtraction, was done on ImageJ software. ER-LAR-GECO and mito-carmine imaging was performed 48-hours following transfection in H9c2 cells and 18-hours after FSK-I stimulation (additional details are included in figure legends). Calcium imaging in ventricular myocytes was done 48-hours after viral transduction. ATeam 1.03 was imaged with a CFP and FRET (CFP-YFP) cube-set on an Olympus IX70 inverted microscope with QImaging. Images were analyzed and quantified using ImageJ software.
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