Embryos were dissected at suitable time points and fixed in 4% PFA/PBS (pH 7.4) at 4 °C O/N then rinsed with PBS at RT several times. Urinary tracts were dissected under the microscope, soaked in PBS-BB (1% BSA, 0.2% skim milk, 0.3% Triton X-100 and 1 × PBS) to block and incubated O/N at 4 °C on a shaker. Tissues were incubated with primary antibody solution diluted in PBS-BB at 4 °C O/N, then washed with PBS-Tr (0.3% Triton X-100,1x PBS) twice at RT for 1–2 h and once at 4 °C O/N on a shaker. Tissues were incubated with secondary antibody diluted in PBS-BB O/N at 4 °C, washed several times in PBS-Tr at RT and imaged in 1 × PBS on a Nikon C1 confocal system with NIS Elements software (Nikon Instruments Inc., America).
C1 confocal system
Manufactured by Nikon
Sourced in Japan, United States
The Nikon C1 confocal system is a high-performance microscope designed for advanced imaging applications. It utilizes laser scanning technology to provide optical sectioning, enabling the capture of detailed, three-dimensional images of samples. The C1 confocal system is capable of delivering high-resolution, low-noise images with excellent contrast and clarity.
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Lab products found in correlation
Ez c1, by Nikon (6 mentions)
C1 plus microscope, by Nikon (3 mentions)
5 ethynyl 2 deoxyuridine (edu), by Thermo Fisher Scientific (3 mentions)
Nis elements software, by Nikon (2 mentions)
Ez c1 3.60 freeviewer software, by Nikon (2 mentions)
Eclipse e800 microscope, by Nikon (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
E800 fluorescent microscope, by Nikon (2 mentions)
Te2000 inverted microscope, by Nikon (1 mentions)
Elements software, by Nikon (1 mentions)
Mowiol mounting media, by Merck Group (1 mentions)
Anti abca1 antibody, by Novus Biologicals (1 mentions)
Goat anti rabbit h l alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Goat serum, by Merck Group (1 mentions)
Ab187149, by Abcam (1 mentions)
Alexa fluor 546, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Celltracker orange, by Thermo Fisher Scientific (1 mentions)
Imagej, by Nikon (1 mentions)
37 protocols using c1 confocal system
1
Immunofluorescence Imaging of Embryonic Urinary Tracts
2
Quantifying Lysosomal Dextran Uptake and Exocytosis
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Cells (6 × 104) were incubated for 2 hours with Alexa Fluor 594–conjugated dextran (0.5 mg/ml; molecular weight, 10,000; anionic) (Life Technologies) to allow for complete endocytosis followed by redistribution and accumulation of the compound in the lysosomes (58 (link)). Cells were washed four times with phosphate-buffered saline (PBS), and the medium was changed. After 1 hour, the medium was spun down and exocytosed. Alexa Fluor 594–dextran fluorescence was measured (excitation, 585/10 nm; emission, 615/10 nm). AFU were normalized to protein concentration.
For dextran uptake, cells were incubated with Alexa Fluor 594–conjugated dextran (0.5 mg/ml; molecular weight, 10,000). After 30 min, cells were washed twice with PBS and imaged with a Nikon C1 confocal system mounted on a Nikon TE2000 inverted microscope fitted with an incubator chamber for humidity, CO2, and temperature control. Seven to 10 images were acquired for each group of cells. The fluorescence intensity (in arbitrary units) of the red channel was determined for individual cells; the mean + SD are shown in the graph.
For dextran uptake, cells were incubated with Alexa Fluor 594–conjugated dextran (0.5 mg/ml; molecular weight, 10,000). After 30 min, cells were washed twice with PBS and imaged with a Nikon C1 confocal system mounted on a Nikon TE2000 inverted microscope fitted with an incubator chamber for humidity, CO2, and temperature control. Seven to 10 images were acquired for each group of cells. The fluorescence intensity (in arbitrary units) of the red channel was determined for individual cells; the mean + SD are shown in the graph.
3
Cell Wall Imaging via Confocal Microscopy
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Semi-thin (300 nm) sectioned samples were positioned on glass microscope slides and stained with 0.1% acriflavine for confocal scanning laser microscopy of cell walls. Images were captured using a Nikon C1 Plus microscope (Nikon, Tokyo, Japan), equipped with the Nikon C1 confocal system with four lasers (403 nm, 561 nm, 643 nm, and Argon tunable 458/477/488/515 nm), and operated via Nikon’s EZ-C1 software.
4
Confocal Imaging of Agave Leaf Anatomy
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A confocal laser scanning microscope (Nikon ECLIPSE E800 microscope equipped with the Nikon C1 confocal system) (Nikon Instruments Inc. Melville, NY, USA) was used for imaging the fresh cut transverse section of agave leaf. Images of white light and auto-fluorescence were excited by a 488 nm laser and detected by 515/30 nm emission filter. All images were recorded at a resolution of 4048 × 3027 pixels.
5
Preparation of Scleral Flat Mounts
6
Localization of ABCA1 in Trophoblast Monolayer
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In addition to determining the biophysical and biochemical properties of the trophoblast monolayer, the localization of ABCA1 was analysed using confocal microscopy, as previously described [46 (link)]. In brief, the confluent trophoblast monolayers grown on Transwell® membranes were first fixed in methanol at −20 °C for 30 min, and then washed in 0.1 M glycine (Merck, Darmstadt, Germany) for 15 min. After blocking with a cocktail of 2% bovine serum albumin (Sigma, Saint Louis, MO, USA) and 4% goat serum (Sigma, Saint Louis, MO, USA) for 1 h at room temperature, they were incubated with anti-ABCA1 antibody (Novus Biologicals, Littleton, CO, USA) overnight at 4 °C. After washing, the mixture of goat anti-rabbit (H + L) Alexa Fluor 488 (Invitrogen, Eugene, OR, USA) with 4’,6-diamidino-2-phenylindole (DAPI; Sigma, Saint Louis, MO, USA) was added for 1 h at room temperature in the dark. Membranes were mounted on glass slides using Mowiol mounting media (Sigma, Saint Louis, MO, USA). The immunofluorescent staining was examined using a Nikon C1 confocal system (Nikon, Tokyo, Japan) equipped with a 40× S Fluor oil-immersion objective. Images were acquired and optimized with EZ-C1 software (Nikon, Tokyo, Japan).
7
Immunofluorescence Imaging of SMYD3 and MYC
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CHLA01 cells were fixed with freshly made 4% paraformaldehyde and immuno-stained with anti-SMYD3 (ab187149, 1:250, Abcam, Cambridge, UK) and anti-MYC tag (ab32, 1:200, Abcam) antibodies. Primary antibody-antigen complexes were visualized using anti-mouse Alexa Fluor 488 and anti-rabbit Alexa Fluor 546 secondary antibodies (Molecular Probes, Eugene, OR, USA; 1:500). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Stained sections were imaged using a confocal laser-scanning microscope (Nikon C1 confocal system, Nikon Corp, Tokyo, Japan). The acquired images were processed using NIS (Nikon) and analyzed with Image J software (https://imagej.nih.gov/ij/ ).
8
Imaging Cervical Cells with Gonorrhea
9
Confocal Microscopy for Multicolor Imaging
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Images were captured using a Nikon C1 Plus microscope (from Nikon in Tokyo, Japan), equipped with the Nikon C1 confocal system and four lasers (403 nm, 561 nm, 643 nm, and Argon tunable 458/477/488/515 nm), and operated via Nikon’s EZ-C1 software. The 435–465-nm filter was also used to detect autofluorescence. Each optical section of each channel series was scanned twice using Nikon EZ-C1 Average. For all images shown, a series of optical sections was collected, and a subset of this series was used to project the images using either Nikon EZ-C1’s Volume Render, Maximum function or ImageJ’s 3D Projection, Max function. ImageJ (from the National Institutes of Health in Bethesda, MD, United States) was used to open projected images, separate and combine color channels, and adjust contrast and brightness of images.
10
Quantitative Imaging Protocol for Protein Expression
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All slides were scanned and imaged by Vectra Polaris (Perkin Elmer) and Nikon C1 confocal system (Tokyo, Japan). All images were analyzed by ImageJ software (NIH, Bethesda, MD, USA). Since there was much deviation, we presented the results as log10 expression/mm2 based on the value we got from ImageJ.
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