For EEA1/LAMP1 antibody with Stabilin-1 or -2 detection, a simultaneous incubation method was used. For simultaneous multicolor staining of EEA1 or LAMP1 with Stabilin-1 or -2, first, cells were incubated with both the primary antibodies in 1× PBS+1% (0.1 g) BSA+0.01% Saponin in a humidified chamber for 1 h at room temperature, followed by washing with PBS thrice (5 min/wash). Next, cells were incubated with both the secondary antibodies in 1× PBS+1% BSA+0.01% Saponin in a humidified chamber for 1 h at room temperature. Next, cells were washed thrice with PBS and then mounted on a coverslip using antifade mounting media (Southern Biotech, catalog #0100-0), and slides were imaged using confocal laser scanning microscopy (CLSM) on a Nikon A1R Ti2 inverted fluorescent microscope using Nikon Elements. A Plan Apo VC 60×/1.40 oil immersion lens was used with a 2× digital zoom for a total magnification of 1,200×. Laser lines used on CLSM were as follows: 405 (nucleus), 488 laser (Stabilin-1/2), 560 laser line (PS-ASO), and 640 laser line (vesicle). Z steps were acquired between 0.7 and 1 μm using channel series/sequence mode. Image analysis was carried out using the EzColocalization plugin in ImageJ.
A1r ti2
Manufactured by Nikon
Sourced in Japan, United States
The Nikon A1R Ti2 is a high-performance confocal microscope designed for advanced imaging applications. It features a resonant scanner for fast image acquisition and a titanium-based frame for enhanced stability and durability. The A1R Ti2 provides high-resolution, multicolor imaging capabilities to support various research and clinical applications.
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Lab products found in correlation
4 protocols using a1r ti2
1
Multicolor Immunofluorescence Staining of Endosomal Markers
2
Imaging Mitochondrial and Lysosomal Dynamics
3
Characterization of Carotenoid Microstructures
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The microstructures of the extracts, and bioaccessible fractions obtained after simulated digestion were characterized using a confocal scanning fluorescence microscope (Nikon A1R–Ti2, Nikon, Tokyo, Japan). Carotenoids naturally exhibit fluorescence, therefore no staining procedure was applied before confocal analysis. A small aliquot (5 μL) of each sample was placed on the microscope slide and covered with a cover slip. The fluorescence signal was excited using laser source with wavelength of 488 nm and the images were recorded at wavelength of 525 nm. All images were captured using a 60× oil immersion objective lens. The hexane- and SC-CO2-extracted oleoresins and their oil fractions were analyzed at a lower laser power compared to their bioaccessible fractions due to the high concentration of lycopene in the extracts. (all-E)-lycopene standard and tomato seed oil were analyzed under confocal microscopy at the same settings as positive and negative controls, respectively.
4
3D Fluorescence Imaging of Corn Syrup Droplet
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A confocal fluorescence microscope (A1R-Ti2, Nikon, Melville, NY, USA) was used for imaging the drop. A transparent container filled with mineral oil was placed on the stage of the microscope. The Hele-Shaw cell was submerged in the oil and then fixed by placing weights on it. Then, the corn syrup solution with the fluorescent dye was injected through the injection hole of the Hele-Shaw cell using a syringe and tubing. By pushing the plunger of the syringe manually, a corn syrup drop was grown in the Hele-Shaw cell slowly. The injection was stopped when the drop had grown to an approximate diameter of 3.5 mm, and then the device stood still for two minutes to stabilize the drop.
The drop was imaged by using the microscope with a 4× objective lens (Plan Apo λ, NA = 0.2) and a z-step size of 20 μm. The excitation and emission wavelengths were 486 nm and 525 nm, respectively. Since the vertical scanning range was 1.1–1.5 mm, which included the entire gap height of the Hele-Shaw cell, 55–70 layers of 2D fluorescent images were obtained. The size of 2D images was 1024 pixel × 1024 pixel, and the pixel size was 3.11 µm/pixel. 2D images were re-constructed into a 3D image using the microscope software (NIS Elements Viewer, Nikon, Melville, NY, USA), and then the 3D image was saved as a gray-scale tiff image file. A bright-field image of the top view of the drop was also captured.
The drop was imaged by using the microscope with a 4× objective lens (Plan Apo λ, NA = 0.2) and a z-step size of 20 μm. The excitation and emission wavelengths were 486 nm and 525 nm, respectively. Since the vertical scanning range was 1.1–1.5 mm, which included the entire gap height of the Hele-Shaw cell, 55–70 layers of 2D fluorescent images were obtained. The size of 2D images was 1024 pixel × 1024 pixel, and the pixel size was 3.11 µm/pixel. 2D images were re-constructed into a 3D image using the microscope software (NIS Elements Viewer, Nikon, Melville, NY, USA), and then the 3D image was saved as a gray-scale tiff image file. A bright-field image of the top view of the drop was also captured.
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