We used magnesium-Fluo-4-acetoxymethylester (mag-Fluo-4/AM) (M14206, Thermo Fisher Scientific, Eugene, OR, USA), which demonstrates increased fluorescence upon Ca2+ binding, to indicate SR free Ca2+, as per Park et al. (2000) [26 (link)]. Briefly, after washing samples twice with fresh PBS, dye (5 mM mag-Fluo-4/AM) was slowly added along the sides of the single muscle fibers, followed by incubation in the dark at 37 °C for 30 min. After incubation, the glass slide-mounted mag-Fluo-4/AM-loaded fibers were washed with fresh PBS three times (20 s/time, 1-min process). The slide was then quickly placed on the microscope stage, with the fibers focused in the bright field (20-s process) and scanned via laser confocal microscopy in combination with an Olympus FV10-ASW system (Japan) under 488-nm krypton/argon laser illumination, with fluorescence detected at 526 nm. Analysis and statistical methods were similar to those used for the measurement of cytoplasmic Ca2+ mentioned above.
Fv10 asw system
Manufactured by Olympus
Sourced in Japan
The FV10-ASW system is a confocal laser scanning microscope designed for high-resolution imaging. It features a flexible and modular architecture, allowing for customization to meet various research needs. The system provides advanced imaging capabilities, but a detailed unbiased description of its core function is not available.
Automatically generated - may contain errors
Lab products found in correlation
Fluoview fv1000, by Olympus (2 mentions)
Mag fluo 4 am, by Thermo Fisher Scientific (1 mentions)
Fluo 3 am, by Olympus (1 mentions)
Fluo 3 am, by Thermo Fisher Scientific (1 mentions)
Fv10 asw 4.0 viewer, by Olympus (1 mentions)
μ slide 8 well, by Ibidi (1 mentions)
Rhod 2 am, by Thermo Fisher Scientific (1 mentions)
Live dead baclight viability kit, by Thermo Fisher Scientific (1 mentions)
Hoechst 33258, by Thermo Fisher Scientific (1 mentions)
Anti cathepsin d, by Santa Cruz Biotechnology (1 mentions)
Anti cx43, by Santa Cruz Biotechnology (1 mentions)
Anti lc3β, by Santa Cruz Biotechnology (1 mentions)
Anti c9, by Santa Cruz Biotechnology (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Image pro plus 5, by Media Cybernetics (1 mentions)
9 protocols using fv10 asw system
1
Measuring Sarcoplasmic Reticulum Calcium
2
Measuring Intracellular Calcium in Muscle Fibers
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Fluo-3-acetoxymethylester (Fluo-3/AM) (Invitrogen, Carlsbad, USA), which exhibited an increase in fluorescence upon binding Ca2+, was used to indicate cytosolic free Ca2+ as previous described50 (link). Briefly, the single muscle fibers were incubated with Fluo-3/AM in a concentration of 5 mM for 30 min at 37 °C. After incubation, the Fluo-3/AM-loaded muscle fibers on a glass slide were washed with fresh PBS and then scanned under a laser confocal microscope equipped with the Olympus FV10-ASW system (Olympus, FV10-MCPSU, Japan) by illuminating with a krypton/argon laser at 488 nm emitted light and capturing the emitting fluorescence at 526 nm. The fluorescence intensity was used to indicate the change of intracellular Ca2+ in muscle fibers. Quantification analysis of the fluorescence intensity was performed with the NIH Image software (Image-proplus 6.0).
3
Visualizing MSNs in Biofilms
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The 1-day-old biofilms were prepared in the chambered cover glass (μ Slide 8 well; ibidi GmbH, Munich, Germany), as described previously. Before the treatment, the RITC-S-/RITC-W-MSNs were dispersed in BHI broth or YNB containing 100 mM glucose by sonication for 10 minutes. Then the RITC-S-/RITC-W-MSNs dispersions were added to the biofilms followed by 24-hour incubation. The treated biofilms were subsequently washed in PBS once to remove the free nanoparticles and then stained with SYTO-9 (Live/Dead BacLightTM Viability Kit; Thermo Fisher Scientific) for 30 minutes in the dark at room temperature. The images of the fluorescent MSNs in the biofilms were visualized by an Olympus FLUOVIEW FV 1000 confocal scanning laser microscope with FV-10 ASW system (Olympus Corporation, Tokyo, Japan). This microscope equipped with 543 nm HeNe laser and 488 nm Argon laser were used to obtain the red signals from RITC-labeled MSNs and green signals of SYTO-9, respectively. The image analysis was performed with FV10-ASW 4.0 Viewer (Olympus Corporation) and ImageJ. Briefly, the biofilm images with multiple layers were projected over z-axis and transformed into tiff format. The images of red channel, which represents the distribution of RITC-MSNs, were opened with ImageJ to calculate the number of particles, average size, and percentage of RITC-MSN area.
4
Measuring Mitochondrial Calcium Levels
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We used Rhod-2/AM (R1244, Thermo Fisher Scientific, USA), which demonstrates increased fluorescence upon Ca2+ binding in the mitochondria, to determine mitochondrial free Ca2+ [27 (link)]. Briefly, after washing samples twice with fresh PBS, dye (5 µM Rhod-2/AM) was slowly added along the sides of the single muscle fibers, followed by incubation in the dark at 37 °C for 30 min. After incubation, the glass slide-mounted Rhod-2/AM-loaded fibers were washed with fresh PBS three times (20 s/time, 1-min process). The slide was then quickly placed on the microscope stage, and the fibers were focused in the bright field (20-s process) and scanned via laser confocal microscopy in combination with an Olympus FV10-ASW system (Japan) under 594-nm krypton/argon laser illumination, with fluorescence detected at 618 nm. Analysis and statistical methods were similar to those used for the measurement of cytoplasmic Ca2+ mentioned above.
5
Biofilm Viability Assessment via Confocal Microscopy
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All anti-biofilm setups were identical to previous sections except that the bacteria were cultured in ibidi GmbH μ-Slide 8-well chambers with a polymer coverslip (Munich, Germany). After removing the treatments, the biofilms were stained with SYTO9 and propidium iodide from Live/Dead BacLight™ viability kit (Thermo Fisher Scientific) for 30 min at room temperature, and the biofilm viabilities were assessed using a confocal scanning laser microscope equipped with 543 nm HeNe laser and 488 nm Argon laser (Olympus FLUOVIEW FV 1000 with an FV-10 ASW system; Tokyo, Japan). The as-obtained fluorescence images were analyzed by ImageJ (Version 1.8.0, National Institutes of Health, USA).
6
Adenovirus-mediated Autophagy Visualization
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Cells were seeded in a 24-well plate (5x104 cells) and infected with RFP-GFP-LC3-labeled adenovirus (Hanbio Biotechnology Co., Ltd.) at a multiplicity of infection of 50. After incubating for 24 h in an incubator at 37˚C supplied with 5% CO2, the supernatant of the medium containing the virus solution was discarded and replaced with complete medium (MEM medium containing 10% FBS for FaDu cells and BEBM medium containing 10% FBS for FD-LSC-1 cells). After incubating for 24 h in an incubator at 37˚C, images were taken using a laser-scanning confocal microscope equipped with the FV10-ASW system (magnification, ⅹ400; Olympus Corporation).
7
Visualizing Myocardial Infarct Using Evans Blue
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Five minutes after reperfusion, the hearts were perfused with 0.01% Evans blue dye for 5 min and then harvested. A 4-μm thick frozen section was prepared and washed with acetone. The tissue sections were examined with a laser-scanning confocal microscope, and the images were scanned with an FV-10-ASW system (Olympus FV1000, Tokyo, Japan). The excitation and emission wavelengths were 543 and 590 nm, respectively. A histologist who was unaware of the group assignment examined all sections, and the percentage of positive cells was calculated as described previously10 (link),36 (link).
8
GLUT4 Localization in Rat Soleus Muscle
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Soleus muscle samples from the rats with or without insulin stimulation were cut from the middle of the muscle belly and frozen with optimum cutting temperature compound and isopentane in liquid nitrogen. Frozen 10 μm-thick muscle cross sections were obtained in a freezing cryostat at −20°C. The sections were air dried at room temperature, fixed in ice-cold acetone for 30 min, permeabilized in the phosphate buffered saline (PBS; 135 mM NaCl, 10 mM sodium phosphate pH 7.0) including 0.1% Triton X-100 for 30 min, and blocked in 1% bovine serum albumin (BSA) in PBS for 60 min at room temperature. Sections were incubated with anti-GLUT4 (1 : 100 dilution) at 4°C overnight. The slides were rinsed twice in PBS and incubated with rhodamine-labeled goat-anti-rabbit IgG (1 : 400 dilution; Invitrogen, Carlsbad, USA) for 60 min. Stained sections were observed using a laser-scanning confocal microscope equipped with the FV10-ASW system (Olympus FV1000). The rhodamine-labeled signals were visualized at 519 nm. Images were acquired at a 60x water objective. Optical densitometry analysis of GLUT4 was performed using Olympus Fluoview image analysis software (Olympus Co., Ltd.). Nuclei were identified by the Hoechst 33258 (1 : 100 dilution; Invitrogen) staining.
9
Immunofluorescent Analysis of Myocardial Tissue
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Hearts were removed after ischemia-reperfusion, fixed in 4% paraformaldehyde, embedded into OCT compound, quickly frozen at -80°C, and then processed for cryosectioning (5 µm thickness). Ten sections were prepared at 10 different transversal levels at the site of tissue necrosis, equally distributed from base to apex.
After the cryosections were fixed with acetone at 4°C for 30 min, the sections were stained with anti-Cx43 (1:200; Santa Cruz Biotechnology, Inc., Santa Cruz, Calif., USA) and anti-C9 (1:100; Santa Cruz Biotechnology, Inc., Santa Cruz, Calif., USA), anti-Lc3-β (1:50; Santa Cruz Biotechnology, Inc., Santa Cruz, Calif., USA) or anti-cathepsin D (1:50; Santa Cruz Biotechnology, Inc., Santa Cruz, Calif., USA) at D r a f t 4°C overnight. This procedure was followed by a 60 min incubation at room temperature with Alexa Fluor 488 goat anti-mouse IgG or tetramethylrhodamine goat anti-rabbit IgG second antibody. Hoechst 33342 (5 µg/mL, Molecular Probe) was used to label the nuclei. The sections were examined using a laser-scanning confocal microscope equipped with the FV10-ASW system (Olympus FV1000). The images were analyzed using Image-Pro Plus 5.0 software (Media Cybernetics Inc., Rockville, Md., USA)
After the cryosections were fixed with acetone at 4°C for 30 min, the sections were stained with anti-Cx43 (1:200; Santa Cruz Biotechnology, Inc., Santa Cruz, Calif., USA) and anti-C9 (1:100; Santa Cruz Biotechnology, Inc., Santa Cruz, Calif., USA), anti-Lc3-β (1:50; Santa Cruz Biotechnology, Inc., Santa Cruz, Calif., USA) or anti-cathepsin D (1:50; Santa Cruz Biotechnology, Inc., Santa Cruz, Calif., USA) at D r a f t 4°C overnight. This procedure was followed by a 60 min incubation at room temperature with Alexa Fluor 488 goat anti-mouse IgG or tetramethylrhodamine goat anti-rabbit IgG second antibody. Hoechst 33342 (5 µg/mL, Molecular Probe) was used to label the nuclei. The sections were examined using a laser-scanning confocal microscope equipped with the FV10-ASW system (Olympus FV1000). The images were analyzed using Image-Pro Plus 5.0 software (Media Cybernetics Inc., Rockville, Md., USA)
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