Cells were grown on 13mm glass coverslips (Menzel, Braunschweig, Germany) in 24-well plastic cell culture plates (NunclonTM) and fixed with 4% paraformaldehyde. After incubation with the primary antibody overnight and with the appropriate secondary antibody for 1 h, Hoechst-33342 (1 µg/ml) was added for 10 min prior to the final washing step. Coverslips were mounted on glass slides with Fluorsave reagent (Calbiochem/Millipore/Darmstadt/Germany). For visualisation, an Olympus IX81 inverted epifluorescence microscope (Hamburg, Germany) was used. For image processing, Image J open-source software was used.
Ix81 inverted epifluorescence microscope
Manufactured by Olympus
Sourced in Japan
The Olympus IX81 inverted epifluorescence microscope is a laboratory equipment designed for advanced imaging and analysis. It features an inverted optical configuration, which allows for the observation of samples from below. The IX81 is capable of epifluorescence microscopy, enabling the visualization of fluorescently labeled samples. The microscope's core function is to provide high-quality, detailed images of specimens for research and analysis purposes.
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Lab products found in correlation
Uplansapo, by Olympus (4 mentions)
Cell imaging medium, by Thermo Fisher Scientific (2 mentions)
Axioskop 2 plus microscope, by Zeiss (2 mentions)
In situ cell death detection kit, by Roche (1 mentions)
Gfp filter cube, by IDEX Corporation (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Oregon green 488 bapta 1 am, by Thermo Fisher Scientific (1 mentions)
Arrayscan vti hcs reader, by Thermo Fisher Scientific (1 mentions)
Vhcs scan, by Thermo Fisher Scientific (1 mentions)
Cellr software, by Olympus (1 mentions)
Orca er ccd camera, by Hamamatsu Photonics (1 mentions)
Metamorph software, by Molecular Devices (1 mentions)
Ikon m ccd camera, by Oxford Instruments (1 mentions)
Metamorph, by Molecular Devices (1 mentions)
Xcite 120q fluorescence lamp, by Excelitas (1 mentions)
Leibovitz s l 15 cell culture media, by Thermo Fisher Scientific (1 mentions)
18 protocols using ix81 inverted epifluorescence microscope
1
Immunofluorescence Staining of Cells
2
TUNEL Assay for Apoptosis Detection
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Apoptotic markers were analyzed on tissue sections by a Terminal deoxynucleotidyl transferase dUTP Nick End Labeling (TUNEL) assay using the In Situ Cell Death Detection Kit (Roche, 000000011684795910). Images were taken with an Olympus IX81 inverted epifluorescence microscope. Postacquisition analysis of the pictures was performed using the ImageJ software.
3
Imaging and Photostability of NIR FPs
4
Calcium Flux Imaging of Differentiated iSNs
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Differentiated mature iSNs were loaded with Fluo‐4‐AM fluorescence dye (Invitrogen) by incubating at room temperature for 1 hour followed by 45 minutes period for de‐esterification. Cells were washed and incubated in Hanks' balanced salt solution, supplemented with 25 mM HEPES buffer and 5.5 mM glucose. Calcium flux was monitored using an Olympus IX81 inverted epi‐fluorescence microscope (Olympus, Markham, ON, Canada) coupled to a xenon arc lamp (EXFO, Quebec, QC, Canada). Indicated agonists, α,β‐meATP and capsaicin, were diluted in the aforementioned solution for a final stimulation concentration of 30 μM for α,β‐meATP and 1 μM for capsaicin. Fluorescence images were collected using an EMCCD (Electron Multiplying Charged Coupled Device) camera (Photometrics, Tucson, AR, USA) every 2 seconds through a GFP filter cube (Semrock, Rochester, NY, USA). Off‐line analysis of the intensity pattern of Fluo‐4 signal was performed in ImageJ (NIH, Bethesda, MD, USA).
5
Live Cell Fluorescence Imaging
6
Intracellular Ca2+ Imaging in Cells
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Ca2+ recordings were performed as described previously (Langeslag, Clark, Moolenaar, van Leeuwen, & Jalink, 2007 ; Visser et al., 2013 ). In short, cells were grown in 24 wells plate and incubated with Oregon Green 488 BAPTA‐1‐AM (Molecular Probes, USA) followed by further incubation in 2 ml DMEM F/12. The plate was mounted on an IX81 inverted epifluorescence microscope (Olympus, Japan). Recordings were made at 37°C in 5% CO2 and 80% humidity. Excitation of Oregon Green‐488 was performed at 480/25 nm and emission was detected between 502 and 538 nm. All Ca2+ recordings were normalized by setting the response to ionomycin (Sigma‐Aldrich, USA) at 100%.
7
Microscopic Imaging of Sample
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Darkfield images were taken with a Zeiss Axioskop 2 plus microscope equipped with a black-white CCD camera. All images were taken at 60X magnification under the same lighting conditions using an Olympus IX-81 inverted epifluorescence microscope.
8
Imaging N2a-hApoE Cellular Morphology
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Cellular appearances were evaluated and compared using phase contrast imaging. N2a-hApoE cells were seeded in 35 mm glass-bottom poly-D-lysine pre-coated culture dishes (MatTek Corporation, Ashland, MA, USA). Cells were maintained for 4 days and were replaced with fresh medium every 2 days. Phase contrast images were acquired using an Olympus IX81 inverted epifluorescence microscope equipped with a cage incubator for maintaining precise temperature, humidity and CO2 control. Slidebook Software Version 6.0 was used for microscope control, image acquisition, image processing, and data analysis (Intelligent Imaging Innovations, Inc., Denver, CO, USA). Images were acquired using a 40× phase contrast magnification on day 4 under 37 °C and 5% CO2.
9
Live Cell Imaging and Analysis
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Labeling live cells was performed with 1 µM calcein-AM/1 µg/ml H-33342 for 30 min at 37 °C. Images were collected in two different fluorescent channels using an automated microscope (Array-Scan VTI HCS Reader, Thermo Fisher, PA, USA) with high content imaging software (vHCS SCAN, Thermo Fisher, PA, USA). For visualization, an Olympus IX81 inverted epifluorescence microscope with a 20× objective was used. Nuclei were automatically identified in channel 1 (365 ± 50/461 ± 15 nm) as objects according to their size, area, shape, and intensity. The calcein signal was detected in channel 2 (475 ± 40/525 ± 15 nm). An algorithm quantified all calcein positive cells as viable and nuclei stained by H-33342 only as “non viable” cells.
For quantification of the neurite area of d3 cells a well-established algorithm was applied (Stiegler et al. 2011 (link)). For d6 LUHMES, cells were fixed and stained for β-III-tubulin and H-33342, and then the same algorithm was applied.
For quantification of the neurite area of d3 cells a well-established algorithm was applied (Stiegler et al. 2011 (link)). For d6 LUHMES, cells were fixed and stained for β-III-tubulin and H-33342, and then the same algorithm was applied.
10
Live Cell Fluorescence Imaging
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Live HeLa, HEK293, NIH3T3 and COS-1 cells were imaged with an Olympus IX81 inverted epifluorescence microscope 72 h after the transfection. The microscope was equipped with a 200-W metal halide arc lamp (Lumen220PRO, Prior), a 60 × 1.35 numerical aperture (NA) oil objective lens (UPlanSApo, Olympus) and an opiMOS sCMOS camera (QImaging). During imaging, cells were incubated in a cell imaging medium (Life Technologies-Invitrogen) at room temperature. The microscope was operated with a SlideBook v.6.0.8 software (Intelligent Imaging Innovations).
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