The intracellular localization of DAR-4M micelles was detected using LysoTracker Green DND-26 (Life Technologies Japan.,Ltd., Tokyo, Japan) according to the product manual. Briefly, RAW264.7 cells were seeded on glass bottom dish (Matsunami Glass Ind.,Ltd., Osaka, Japan) at a density of 1 × 105 cells/cm2 and cultured in 1 ml of DMEM medium with 10 vol% FCS for 24 h. The medium was exchanged to DMEM containing 100 ng/ml of LPS, and incubated further for 24 h. Next, the medium was exchanged to FCS-free DMEM containing 75 nM of LysoTracker Green DND-26, and then, DAR-4M micelles were added to each dish. After 1 h incubation, cells were washed with PBS. The cells were viewed on a Nikon ECLIPSE 90i confocal laser scanning microscope (Nikon Corp., Tokyo, Japan).
Eclipse 90i confocal laser scanning microscope
Manufactured by Nikon
Sourced in Japan
The Eclipse 90i confocal laser scanning microscope is a high-performance laboratory instrument designed for advanced imaging applications. It utilizes laser technology to capture detailed, high-resolution images of samples at the microscopic level. The core function of this microscope is to provide researchers with a powerful tool for investigating and analyzing various specimens with exceptional clarity and precision.
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Lab products found in correlation
Glass bottom dish, by Matsunami (2 mentions)
Lysotracker green dnd 26, by Thermo Fisher Scientific (1 mentions)
24 well multi dish culture plate, by Corning (1 mentions)
Leadmium green am solution, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti rabbit secondary antibody, by Thermo Fisher Scientific (1 mentions)
5 protocols using eclipse 90i confocal laser scanning microscope
1
Intracellular Localization of DAR-4M Micelles
2
Fluorescent Intensity of RAW264.7 Cells
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RAW264.7 cells were seeded on glass bottom dish (Matsunami Glass Ind.,Ltd., Osaka, Japan) at a density of 1 × 105 cells/cm2 and cultured in 1 ml of DMEM medium with 10 vol% FCS for 24 h. The medium was exchanged to DMEM containing 100 ng/ml of LPS, and incubated further for 24 h. Next, the medium was exchanged to FCS-free DMEM, and then, free DAR-4M, DSPE-g-gelatin, the mixture of free DAR-4M and DSPE-g-gelatin, or DAR-4M micelles were added to each dish. After 1 h incubation, cells were washed with PBS. The cells were viewed on a Nikon ECLIPSE 90i confocal laser scanning microscope (Nikon Corp., Tokyo, Japan).
To evaluate the fluorescent intensity of cells incubated for 1 h with free DAR-4M, DSPE-g-gelatin, the mixture of free DAR-4M and DSPE-g-gelatin, and DAR-4M micelles, RAW264.7 cells were seeded on each well of 24 well multi-dish culture plate (Corning Inc., Corning, NY) at a density of 1 × 105 cells/cm2 cultured in 1 ml of DMEM with 10 vol% FCS for 24 h. Then, the medium was exchanged to FCS-free DMEM, and free DAR-4M, DSPE-g-gelatin, the mixture of free DAR-4M and DSPE-g-gelatin or DAR-4M micelles were added to each well. After 1 h incubation, cells were washed with PBS. The cells were viewed on a Nikon ECLIPSE 90i confocal laser scanning microscope (Nikon Corp., Tokyo, Japan).
To evaluate the fluorescent intensity of cells incubated for 1 h with free DAR-4M, DSPE-g-gelatin, the mixture of free DAR-4M and DSPE-g-gelatin, and DAR-4M micelles, RAW264.7 cells were seeded on each well of 24 well multi-dish culture plate (Corning Inc., Corning, NY) at a density of 1 × 105 cells/cm2 cultured in 1 ml of DMEM with 10 vol% FCS for 24 h. Then, the medium was exchanged to FCS-free DMEM, and free DAR-4M, DSPE-g-gelatin, the mixture of free DAR-4M and DSPE-g-gelatin or DAR-4M micelles were added to each well. After 1 h incubation, cells were washed with PBS. The cells were viewed on a Nikon ECLIPSE 90i confocal laser scanning microscope (Nikon Corp., Tokyo, Japan).
3
Quantifying Lens Cell Proliferation
4
Lead Accumulation Visualization in Salix babylonica
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S. babylonica root tips exposed to different Pb concentrations (0, 1, 10, 50, or 100 μmol/L) for 3, 6, 12, and 24 h were soaked in EDTA solution (Na2-EDTA, 20 mmol/L) and washed with running water for 15 min. Then, root tips were washed with deionized water 3 times. Afterwards, experimental and control roots were stained using the Pb-specific probe Leadmium™ Green AM solution (Molecular Probes, Invitrogen, Carlsbad, CA, USA) for 90 min at 40 °C in the dark following the manufacturer’s instructions to visualize Pb absorption and distribution [55 ]. Intact cells exhibited green fluorescence due to the Pb-specific probe Leadmium™ Green AM solution. Fluorescence density was analyzed using the “Analyze and Measure” function in Image J software to evaluate the Pb distribution in intact roots. Prepared samples were observed using a Nikon Eclipse 90i confocal laser scanning microscope with an exciter at 488 nm and a barrier at 590/50 nm.
5
Immunofluorescence Analysis of Cnn3 in Mouse Eyes
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Embryonic heads (E12.5 and E16.5) and adult P28 day-old mouse eyes, were fixed in 4% buffered paraformaldehyde, cryoprotected, embedded in OCT (optical cutting temperature) compound and 10 μm sections were cut in sagittal plane using Microm HM 550 as described by us earlier9 (link). Cryosections were incubated overnight with CNN3 rabbit polyclonal antibody (details are described in Table S1 ; Supplemental material) as we described earlier9 (link). Following this, sections were washed and incubated with Alexa Fluor® 488 goat anti-rabbit secondary antibody (Invitrogen) before image capture using an Eclipse 90i confocal laser scanning microscope (Nikon Instruments, Inc.)9 (link).
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