Preparation of 3D culture for immunofluorescence imaging was adapted from a procedure previously described by Debnath et al43 (link). Cultures were initially prepared in glass bottom 24 well plates as described above, and grown for the specified duration prior to paraformaldehyde fixation (2%) and permeabilization with 0.5% Triton X-100. The exchange of buffers and staining solutions was accomplished using a micropipetter to minimize disruption of the Matrigel. The anti-E-cadherin primary antibody used in this study was conjugated directly to Alexa Fluor 488 (Cell Signaling, Danvers, MA, USA). After incubation with antibody solution, cells were washed and covered with mounting medium containing DAPI (Prolong Antifade with DAPI, Invitrogen, Carlsbad, CA) to label nuclei. All confocal immunofluorescence images were obtained using a 40× water immersion objective, with pinhole open to 180 μm, on an Olympus FV-1000 confocal microscope (Olympus of America) and configured for simultaneous imaging of DAPI and Alexa Fluor 488 fluorescence emission without spectral bleed through or cross talk between channels.
Alexa fluor 488
Manufactured by Olympus
Sourced in Japan
Alexa Fluor 488 is a fluorescent dye used in various laboratory techniques, such as flow cytometry, fluorescence microscopy, and immunoassays. It has an excitation maximum at 495 nm and an emission maximum at 519 nm, making it suitable for detection with standard fluorescein filter sets. Alexa Fluor 488 is a bright, photostable dye that can be conjugated to a wide range of biomolecules, including antibodies, proteins, and nucleic acids.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Prolong antifade with dapi, by Thermo Fisher Scientific (1 mentions)
Fv1000 confocal microscope, by Olympus (1 mentions)
Alexa fluor 488, by Cell Signaling Technology (1 mentions)
U mniba, by Olympus (1 mentions)
Can get signal immunostain solution b, by Toyobo (1 mentions)
Cube u mwu, by Olympus (1 mentions)
Crystal violet, by Merck Group (1 mentions)
Ckx41 microscope, by Olympus (1 mentions)
Fluoview 1000 ix81, by Olympus (1 mentions)
Crystal violet, by Olympus (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Ix83 inverted microscope, by Olympus (1 mentions)
Cellsens dimension software, by Olympus (1 mentions)
Orca spark digital camera, by Hamamatsu Photonics (1 mentions)
Hoechst 33342, by Olympus (1 mentions)
Phalloidin conjugated with alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
5 protocols using alexa fluor 488
1
3D Immunofluorescence Imaging of E-cadherin
2
Fluorescence Microscopy Observation of GFP
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For observation of fluorescence of GFP, transformed cells were collected by centrifugation at 200×g for 10 min, and examined by a fluorescence microscope BX-60 (Olympus, Tokyo, Japan), using the cube U-MNIBA (Olympus). When GFP fluorescence was faint, we performed immunofluorescence detection using anti-GFP antibody, essentially according to Nishida et al. (2004) (link). In the reaction with antibody, an anti-GFP monoclonal antibody was diluted to 1/200 with immunoreaction enhancer solution (Can Get Signal immunostain solution B, TOYOBO, Osaka, Japan) and reacted for 1 h. As the secondary antibody, anti-mouse monoclonal antibody labelled with Alexa fluor 488 (Invitrogen, Carlsbad, CA) was diluted to 1/200 with immunoreaction enhancer solution, and reacted for 30 min. The immunostained cells were observed by the fluorescence microscope with the cube U-MNIBA (Olympus) for Alexa fluor 488, the cube U-MWIG (Olympus) for observation of autofluorescence of chlorophyll, and the cube U-MWU (Olympus) for observation of DAPI-stained DNA. Nomarski differential interference image was also recorded. All microscopic images were captured by a digital camera (model DP-70, Olympus).
3
Fluorescent Imaging of Melanoma Cells
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The F-actin and nucleus of melanoma cells were stained as described previously [42 (link)]. Cells were seeded at the density of 3 × 104 cells/well on glass coverslips and next treated with 1 µM AEA or 0.07% v/v DMSO for 24 h. The cells were washed with PBS, fixed with 3.7% PFA/PBS for 30 min at RT, washed with PBS, permeabilized with 0.1% Triton X-100 in PBS for 10 min at RT and again washed with PBS. Then, the actin filaments were stained with a phalloidin labeled with Alexa Fluor 488 (1:300, Life Technologies) in PBS for 30 min at RT and chromatin using the Hoechst 33342 dye (1 μg/mL) in PBS for 15 min at RT. Whole-cell images were collected with the excitation/emission 350 nm/461 nm for Hoechst 33342 and 495 nm/518 nm for phalloidin–Alexa Fluor 488 using the fluorescent microscope (IX71, Olympus).
4
Neurite Quantification in SH-SY5Y Cells
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SH-SY5Y cells were fixed and stained with crystal violet (Sigma-Aldrich), phalloidin conjugated to Alexa-Fluor 488 (Invitrogen), or unstained and imaged under relief contrast conditions on an Olympus CKX41 microscope or Olympus IX81/Fluoview 1000 laser scanning confocal microscope. Images collected after staining with phalloidin (conjugated to Alexa-Fluor 488) and visualized on an Olympus IX81/Fluoview 1000 laser scanning confocal were consistent with neurons visualized by crystal violet staining or relief contrast captured on an Olympus CKX41 microscope. For quantification of neurite length/cell, after crystal violet staining, 10 fields per well were captured with a 4× objective, no contrast, for 6 wells per group. The fields, each 1,400 × 1,050 µm, were preselected in a 2 × 5 array centered in each well, with each field center 3 mm from the center of adjacent fields. Processes were traced and measured automatically with the Neurite Tracer39 (link) macros for ImageJ. Separate experiments were performed and samples were collected under relief contrast (10× objective, 560 × 420 µm fields) for determination of maximum process lengths and percentages of cells with processes by manual tracing (avoiding overlapping cells), assisted and measured with the NeuronJ plugin for ImageJ.
5
Fluorescent Labeling of Actin and Nuclei
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Cells were cultured in Petri dishes until the desired density of cells was obtained, i.e., single cells or cell monolayers. Cells were washed with PBS buffer and fixed by adding a 3.7% paraformaldehyde solution in PBS for 20 minutes. Next, they were washed with PBS three times, and the cell membrane was permeabilized using 0.2% Triton X-100 (Sigma) in PBS at 4° C for 5 minutes. Afterwards, the cells, rinsed with PBS, were labelled with fluorescent dyes in an environment with a minimum light level. Phalloidin conjugated with Alexa Fluor 488 (Invitrogen) was dissolved in PBS buffer (1:200) and used to stain the actin filaments for 30 minutes, while the cell nuclei were stained with Hoechst 33342 (Invitrogen) dissolved in PBS buffer (1:5) for 15 minutes. Afterwards, the samples were washed with PBS buffer. The fluorescent images were recorded using an Olympus IX83 inverted microscope (Olympus, Japan) equipped with a 100 W mercury lamp and a set of filters used to record the emission at 515-545 nm (Alexa Fluor 488) and 461 nm (Hoechst 33342). The images were acquired using the Orca Spark digital camera (Hamamatsu, Japan), providing 2.3 megapixels (1920 pixels per 1200 pixels) images that were recorded using CellSens Dimension software (Olympus).
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