The 5‐μm‐thick LV heart sections were subjected to TUNEL staining and visualized using an Olympus IX81 fluorescent microscope as described previously.30 , 34 Briefly, OCT sections were fixed with 4% paraformaldehyde and then hydrated with PBS at room temperature. Permeabilization was followed by blocking with 1% BSA for 30 minutes followed by incubation with the DNA‐labeling solution for 1 hour at 37°C. Rinsing with buffer was followed by anti‐BrdU incubation at 37°C for 1 hour and counterstaining with propidium iodide for 30 minutes at 37°C; the sections were then mounted with antifade and coverslip and visualized using an Olympus IX81 fluorescent microscope.
Ix81 fluorescent microscope
Manufactured by Olympus
Sourced in Japan, United States
The Olympus IX81 is a high-performance fluorescent microscope designed for advanced biological research. It features a motorized nosepiece, a wide range of objective lenses, and a high-sensitivity camera for capturing detailed fluorescent images. The IX81 is capable of multiple imaging modes, including brightfield, phase contrast, and fluorescence, making it a versatile tool for a variety of applications.
Automatically generated - may contain errors
Lab products found in correlation
Cy2 and cy5 conjugated secondary antibodies, by Jackson ImmunoResearch (2 mentions)
Anti pstat3 antibody, by Cell Signaling Technology (2 mentions)
Lsm 700 confocal microscope, by Zeiss (2 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (2 mentions)
Polybrene, by Merck Group (2 mentions)
Sodium dodecyl sulfate (sds), by Thermo Fisher Scientific (1 mentions)
F8821, by Thermo Fisher Scientific (1 mentions)
Ultracut microtome, by Leica camera (1 mentions)
Anti socs3, by Cell Signaling Technology (1 mentions)
Anti ki67 antibody, by Novus Biologicals (1 mentions)
Glut1, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Hif 1α, by Thermo Fisher Scientific (1 mentions)
Jsm 6700f, by JEOL (1 mentions)
Lambda ls xenon arc lamp, by Sutter Instruments (1 mentions)
Alizarin red solution, by Merck Group (1 mentions)
Coolsnap hq2 camera, by Olympus (1 mentions)
Cytopainter lysosomal staining kit red fluorescence, by Abcam (1 mentions)
Xfect polymer, by Takara Bio (1 mentions)
Normal goat serum (ngs), by Thermo Fisher Scientific (1 mentions)
57 protocols using ix81 fluorescent microscope
1
TUNEL Staining of Cardiac Sections
2
Clonal Analysis of Glioma Stem Cells
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Following pGreenZeo GFAP:GFP or pGreenZeo MAP2:GFP lentivirus transduction, cells were plated at a density of one cell per well in at least four 96-multiwell plates. Wells containing a single neurosphere were further examined using a Olympus IX81 fluorescent microscope for the presence of green fluorescence in at least a subset of the cells comprising the neurosphere. Following clone expansion, flow cytometry was performed to determine the exact percentage of GFP expressing cells. Clones which contained ≤ 5% GFAP:GFP positive cells were designated as GFAP Low or GL. Clones which contained ≥ 75% GFAP:GFP positive cells were designated as GFAP High or GH. Similarly, clones which contained ≤ 5% MAP2:GFP positive cells were designated as MAP2 Low or ML. Clones which contained ≥ 75% MAP2:GFP positive cells were designated as MAP2 High or MH. Parental (non-transduced) GSC lines were used to set baseline fluorescence in all experiments. Because all our clones were derived from single cells and GFP fluorescence was present in at least a subset of cells this procedure ensured that all clones had the reporter cassette integrated in all the cells and that expression of GFP therefore faithfully report GFAP (or MAP2) reporter activity.
3
Cell Traction Force Measurement via Bead Displacement
4
Multimodal Analysis of Intestinal Tumor Samples
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HE staining, IHC, and IF were performed as described previously [19 (link), 20 (link)]. IHC employed the following reagents: anti-Ki67 antibody (Novus Cat. NBP1-40684), anti-SOCS3, and anti-p-Stat3 antibodies (Cell Signaling Technology). IF employed the following reagent: anti-p-Stat3 antibody (Cell Signaling Technology).
For the RNA-FISH assay, the colonic tumor tissues from Apc+/min mice were recovered at necropsy, washed with PBS, fixed in 4% PFA buffer (paraformaldehyde diluted in PBS that had been pre-treated with Diethylpyrocarbonate). The fixed tissues then were embedded in paraffin. After sectioning with an Ultracut microtome (Leica, Bannockburn, IL, USA), the embedded tissues were dehydrated and digested with Protein K, pre-hybridized for 1 h, and hybridized with miR-708 probe overnight at 37 °C. The miR-708 probe consisted of a 6-carboxyfluorescein (FAM)-labeled oligonucleotide with the sequence 5′-FAM-GGGUCGAUCUAACAUUCGAGGAA-FAM-3′; the oligo was purchased from Guangzhou Ribobio Co., Ltd. (Guangzhou, China). After incubation with 4′,6-diamidino-2-phenylindole(DAPI) staining buffer for 10 min, the sections were mounted and then examined with an Olympus IX81 fluorescent microscope.
For the RNA-FISH assay, the colonic tumor tissues from Apc+/min mice were recovered at necropsy, washed with PBS, fixed in 4% PFA buffer (paraformaldehyde diluted in PBS that had been pre-treated with Diethylpyrocarbonate). The fixed tissues then were embedded in paraffin. After sectioning with an Ultracut microtome (Leica, Bannockburn, IL, USA), the embedded tissues were dehydrated and digested with Protein K, pre-hybridized for 1 h, and hybridized with miR-708 probe overnight at 37 °C. The miR-708 probe consisted of a 6-carboxyfluorescein (FAM)-labeled oligonucleotide with the sequence 5′-FAM-GGGUCGAUCUAACAUUCGAGGAA-FAM-3′; the oligo was purchased from Guangzhou Ribobio Co., Ltd. (Guangzhou, China). After incubation with 4′,6-diamidino-2-phenylindole(DAPI) staining buffer for 10 min, the sections were mounted and then examined with an Olympus IX81 fluorescent microscope.
5
Immunofluorescent Staining of Cultured Cells and Brain Tissue
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Coverslips containing 200–300 cells/mm2 were fixed with 4% paraformaldehyde for 20 min followed by treatment with cold ethanol (−20°C) for 5 min and 2 rinses in PBS. The samples were blocked with 3% bovine serum albumin in PBS containing Tween 20 (PBST) for 30 min and incubated in PBST containing 1% bovine serum albumin and goat anti-MAP-2 (1∶50), as described previously [25] (link), [26] (link), [27] (link). After three washes in PBST (15 min each), the slides were further incubated with Cy5 (Jackson ImmunoResearch Laboratories, Inc.). For negative controls, a set of culture slides was incubated under similar conditions without the primary antibodies. The samples were mounted and observed under an Olympus IX81 fluorescent microscope. For tissue staining, brains were kept in 4% paraformaldehyde and 30-µm slices were sectioned in a cryostat followed by immunostaining as described before [28] (link), [29] (link).
6
Evaluating Extracellular Matrix and Hypoxia Markers
7
Neutrophil-Bead Interaction Imaging
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Neutrophils incubated with the antibody tagged fluorescent beads were imaged using Olympus IX81 fluorescent microscope. Five locations in each of the triplicate wells per patient sample were imaged using both bright field and fluorescent microscopy. For the Hoescht 33342 nuclear stain (360–461 nm) blue settings were used, green for the labelled polystyrene beads (460–495 nm), and red for the CellBrite orange membrane dye (540–575 nm). Each location was set using a motorized stage and automated imaging procedure. As such, some images were out of focus and were excluded from analysis.
8
Comprehensive Characterization of Cellular Morphology
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SEM (JSM-6700F, JEOL, Tokyo, Japan) operating at 10 kV was used to characterize cellular morphology. Cellular spheroids were fixed overnight on 2D glass slides with 2.5% glutaraldehyde solution, dehydrated with sequential ethanol solution of 50, 70, 90, 95, and 100%, and then freeze dried. Spheroids were coated by gold sputtering for 120 s and viewed with SEM. UV/Vis spectra were recorded using a UV-3600 UV-Vis-NIR absorption spectrophotometer (Shimadzu Co., Kyoto, Japan), and luminescence spectra with a QuantaMasterTM40 spectrofluorometer in a 1 cm quartz cell at room temperature. The drugs’ luminescence images were acquired using Olympus IX81 fluorescent microscope. The optical density (OD) was measured using Multiskan G0 microplate reader.
9
Fura-2 Calcium Imaging Analysis
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At the time of experimentation, ECs were loaded with fura‐2AM (TefLabs) for 1 hour at 2.5 μmol/L and imaged by acquiring 340/380‐nm fluorescence ratios (R340/380) every 4 seconds, with an Olympus IX81 fluorescent microscope equipped with a Uplan S‐Apo 20× 0.75 NA lens (Olympus), Lambda LS Xenon Arc lamp, Lambda 10‐2 filter wheel shutter controller (both from Sutter Instruments), and RET‐EXi‐F‐M‐12‐C CCD‐camera (QImaging) and controlled with Slidebook 4.2 Imaging software (Olympus). Background fluorescence was determined from a region without cells and was subtracted before ratio determination. R340/380 time‐lapse data acquired from individual cells were baseline subtracted and expressed as an agonist‐induced ΔR340/380. In Mn2+‐quenching experiments,32 (link) cells were additionally excited at 360 nm (D360/10x‐25 filter; Chroma Technology), the isosbestic point for fura‐2, and the resulting 510‐nm fluorescent images were acquired at 3.6‐second intervals.
10
Phagocytosis of Opsonized Staphylococcus Bioparticles
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hTM cells (passage 1) were cultured on gelatin coated
cover-slides in 6-well plates. Meanwhile, Staphylococcus
aureus bioparticles were opsonized according to the
manufacturer’s instructions (Molecular Probes, Eugene,
OR, USA) at 37 °C for 1 h, followed by three PBS
washes. When the cultures reached semiconfluency,
cells were challenged with opsonized Staphylococcus
aureus bioparticle-Alexa Fluor 488 conjugates (Thermo
Scientific) at 50 bioparticles per cell concentration. At 0
and 3 h, cells being incubated at 37 °C were treated with
250 µg/mL Trypan blue (pH 7.2) for 2 min, followed by
three PBS washes to remove free bioparticles. Preparations
were then fixed in 4% paraformaldehyde solution for 20
min, mounted with DAPI-mounting solution, and imaged
using an Olympus IX81 fluorescent microscope.
cover-slides in 6-well plates. Meanwhile, Staphylococcus
aureus bioparticles were opsonized according to the
manufacturer’s instructions (Molecular Probes, Eugene,
OR, USA) at 37 °C for 1 h, followed by three PBS
washes. When the cultures reached semiconfluency,
cells were challenged with opsonized Staphylococcus
aureus bioparticle-Alexa Fluor 488 conjugates (Thermo
Scientific) at 50 bioparticles per cell concentration. At 0
and 3 h, cells being incubated at 37 °C were treated with
250 µg/mL Trypan blue (pH 7.2) for 2 min, followed by
three PBS washes to remove free bioparticles. Preparations
were then fixed in 4% paraformaldehyde solution for 20
min, mounted with DAPI-mounting solution, and imaged
using an Olympus IX81 fluorescent microscope.
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