NeuroBurst Orange Lentivirus (Sartorius, Germany) was used as a genetically-encoded calcium indicator. Virus transduction was performed on day in vitro (DIV) 10 with 3 µl of NeuroBurst. On DIV17, glass coverslips were prepared in an imaging chamber with Tyrode buffer (400 µl) and imaged at 37 °C with an inverted Nikon Ti eclipse epifluorescence microscope (Nikon, Japan). This set-up has an HBO-100 W lamp, a 20X Plan Apo (Nikon, Japan) objective, an IXON X3897 camera (Andor, UK), and an Okolab cage-incubator (Italy). Stimulation was performed with the addition of 400 µl high K+/low Na+ Tyrode buffer (70 mM KCl, 59 mM NaCl, 2 mM CaCl2, 1 mM Mg2Cl, 30 mM D-Glucose and 25 mM HEPES) on top of the initial 400 µl of Tyrode buffer. This solution was not removed till the end of the recording.
Eclipse ti epifluorescence microscope
Manufactured by Nikon
Sourced in Japan, United States
The Eclipse Ti epifluorescence microscope is a high-performance inverted microscope designed for advanced fluorescence imaging. It features a modular design, allowing for customization to suit various research applications. The microscope provides exceptional optical performance, enabling detailed observation and analysis of fluorescently labeled samples.
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Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (8 mentions)
Cellevent caspase 3 7 green detection reagent, by Thermo Fisher Scientific (3 mentions)
Nis elements software, by Nikon (2 mentions)
Donkey serum, by Merck Group (2 mentions)
Triton x, by Merck Group (2 mentions)
Nanog af1997, by R&D Systems (2 mentions)
Hoechst h1399, by Thermo Fisher Scientific (2 mentions)
Tra 1 60 mab4360, by Merck Group (2 mentions)
Ar1 confocal microscope, by Nikon (2 mentions)
Phosphate buffered saline (pbs), by Merck Group (2 mentions)
Ki 67 8d5 mouse monoclonal antibody, by Cell Signaling Technology (2 mentions)
Alexa fluor 488 goat anti mouse secondary antibody, by Thermo Fisher Scientific (2 mentions)
Neuroburst orange lentivirus, by Sartorius (1 mentions)
Plan apo, by Nikon (1 mentions)
Ixon x3897 camera, by Oxford Instruments (1 mentions)
Matlab, by MathWorks (1 mentions)
A1r confocal, by Nikon (1 mentions)
Ixon electron multiplying charge coupled device camera, by Oxford Instruments (1 mentions)
Micromanager, by Nikon (1 mentions)
Formaldehyde, by Merck Group (1 mentions)
37 protocols using eclipse ti epifluorescence microscope
1
Neuronal Calcium Imaging with NeuroBurst
2
Fluorescence Microscopy of Alexa Fluor Dyes
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Samples were imaged on Nikon Ti Eclipse—epifluorescence microscope (Nikon, Düsseldorf, Germany) equipped with a mercury arc lamp, a 40x oil immersion objective (NA 1.3) and standard filter sets (AHF Analysentechnik) to separate the Alexa Fluor dyes.
3
Live Imaging of Synaptic Vesicle Dynamics
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In order to study the synaptic vesicle usage, we took advantage of live staining with an antibody targeting the luminal domain of Synaptotagmin1. At DIV21, coverslips with neurons were placed in a new 12-well plate (Greiner Bio-One) with 300 μl of their own Neurobasal-A medium. Neurons were incubated with 2.5 μg/ml Syt1-Atto647N antibody (105311AT1, Synaptic Systems) for 45 min. Afterward, 16.7 nm anti-mouse secondary nanobody (N2002-At542-S, Nanotag) conjugated to Atto542 was added into the medium and incubated for15 min. Next, the neurons were washed with ice-cold Tyrode's buffer and fixed with 4% PFA. The immunostaining procedure for synaptophysin is described in Immunostaining. In order to see the spontaneous synaptic vesicle fusion, the action potential generation was blocked by adding 5 μm TTX (Tocris Bioscience). In time-series experiments, the uptake assay was performed at different time points of the day and night. For the knocked down conditions, the uptake assay did not have secondary nanobody incubation. Instead, Syt1-Atto647N antibody was incubated for either 15 min or 1 h. An inverted Nikon Ti eclipse epifluorescence microscope (Nikon), which has a 20× Plan Apo (Nikon) objective, an HBO-100W lamp, and an IXON X3897 Andor camera, was used for imaging, and the images were analyzed using MATLAB (The MathWorks).
4
Multimodal Live-cell Microscopy Techniques
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Live-cell microscopy was performed on a Nikon A1R confocal or Nikon Ti Eclipse epifluorescence microscope. Both were fitted with OKO temperature control microscope enclosures (OKO Labs, Pozzuoli, Italy) and stage-top dish incubators for temperature and humidity control (Pathology Devices, Westminster, MD). Both microscopes had motorized stages allowing imaging of multiple fields of view in a well and multiple wells over time. FITC and TRITC images were acquired using a 60×/1.4 numerical aperture Plan APO objective. Dual-excitation and single-emission ratiometric imaging of roGFP2 was done by exciting cells at 405 and 488 nm while collecting emission using a 525/50-nm bandpass filter. For epifluorescence imaging of H2HFF and TRITC, separate images were collected using GFP and Texas red filter sets using an Andor iXon electron-multiplying charge-coupled device camera (Andor Technology, South Windsor, CT). The hardware for confocal imaging was controlled using Nikon Elements, and epifluorescence was controlled using Micromanager (Edelstein et al., 2010 ).
5
Immunofluorescence Staining Protocol
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Cells were fixed with 4% formaldehyde (Sigma) for 15 min at room temperature. Then, cells were washed three times with DPBS and then blocked for 1 h at room temperature with DPBS with 0.4% Triton X-100 and 5% non-fat dry milk (BioRad). Cells were then stained with primary antibodies (Table S3 ) in DPBS with 0.4% Triton X-100 and 5% non-fat dry milk for 2 h. Then cells were washed with DPBS three times and incubated with secondary antibodies (Table S3 ) for 1 h. Cells were washed three times with DPBS. Nuclei were stained with Hoechst 33342 (Thermo Fisher Scientific). A Nikon Ti Eclipse epifluorescence microscope was used for image capture and analysis. Fiji and MATLAB were used for further analyses and quantification.
6
Quantitative Histochemical Analysis of Cellular Enzyme Activity
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Cells were washed three times with PBS and then fixed for 20 minutes at room temperature with 100 μl acetate buffer (110 mM sodium acetate, 50 mM sodium tartrate dibasic dehydrate, 0.28% glacial acetic acid, pH 4.8). Napthol AS-BI Phosphate Substrate (44 mM in 2-ethoxyethanol) was diluted in acetate buffer at a ratio of 1:100, and 100 μl of this solution was added to each well for 1 hour at 37°C. Sodium nitrite (0.58 M) and pararosaniline dye (154 mM in 2M HCl) were combined 1:1 and then diluted in acetate buffer at a ratio of 1:25. The napthol solution was removed from the cells, and 100 μl of the pararosaniline solution was added directly without any washing step. Cells were incubated at 37°C for 5–10 minutes until a red stain developed. The reaction was stopped with three washes in distilled water and then stored in water at 4°C. Bright-field images were obtained using a 4x objective on a Nikon Ti Eclipse epi-fluorescence microscope.
7
Microfluidic PDMS Device Fabrication and Imaging
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The microfluidic PDMS (Sylgard 182, Dow Corning) device was fabricated using standard soft lithography as previously described [44 (link)]. The microfluidic device is a combination of a gradient mixer [45 (link)] and bacterial traps designed with dimensions of 200 μm x 10–50 μm x 1 μm as a H-shaped chemostat (S1 Fig ).
Time-lapse microscopy measurements were conducted on a Nikon Ti-Eclipse epi-fluorescence microscope controlled with NIS-Elements Imaging Software. The microscope was equipped with a sCMOS camera (Zyla, Andor), an automated x-y-stage (Prior Scientific, Cambridge, UK) and an incubator box (Okolab) to maintain an operation temperature of 37°C. All videos were recorded with 40x apochromatic magnification objectives. Every 5 to 20 min, images in phase contrast mode, YFP as well as RFP fluorescence mode (in combination with the appropriate filter sets) were taken for a total run time of up to 20 hours. The exposure times were automatically adjusted.
Time-lapse microscopy measurements were conducted on a Nikon Ti-Eclipse epi-fluorescence microscope controlled with NIS-Elements Imaging Software. The microscope was equipped with a sCMOS camera (Zyla, Andor), an automated x-y-stage (Prior Scientific, Cambridge, UK) and an incubator box (Okolab) to maintain an operation temperature of 37°C. All videos were recorded with 40x apochromatic magnification objectives. Every 5 to 20 min, images in phase contrast mode, YFP as well as RFP fluorescence mode (in combination with the appropriate filter sets) were taken for a total run time of up to 20 hours. The exposure times were automatically adjusted.
8
Time-lapse Imaging of Bacterial Surface Attachment
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Bacterial strains were subcultured in liquid M8 supplemented with glucose, MgSO4, and casamino acids after being cultured overnight in liquid LB at 37C. After reaching an OD600~0.5, cultures were diluted 1:100 into fresh, liquid M8 and then 300ul was used to fill a glass bottom chamber (Cellvis 8 chambered cover glass system). The chamber was then mounted on a Nikon Ti Eclipse epifluorescence microscope in an environmental chamber set to 37C. Images of at least 3 FOV were taken every 5 minutes for the first 8 hours of surface attachment. Images were then analyzed using a python script, which can be found at https://github.com/GeiselBiofilm .
9
Single-Molecule FISH for Sox2 mRNA
10
Imaging Kidney Tissue with smFISH
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Immunostained and smFISH-treated kidney sections were imaged on a Nikon Ti-Eclipse epifluorescence microscope equipped with an Andor iXON Ultra 888 EMCCD camera, using a 100× /1.45 Plan Apo Lambda oil objective (Nikon, Tokyo, Japan) and dedicated, custom-made fluorescence filter sets (Nikon). To cover large areas of the sectioned kidney, images of multiple adjacent areas were taken and combined using the tiling feature of the NIS Elements software (Nikon). For imaging of smFISH signals, z-stacks were collected with distances of 0.3–0.5 μm between planes in four fluorescence channels (GFP, Quasar 570, CAL FLuor Red 610, Quasar 670).
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