All fluorescence images were acquired using a TE-2000 epifluorescence microscope (Nikon) equipped with integrated Perfect-Focus System (PFS), Nikon Plan Apochromat 20× objective lens and Photometrics Cool SNAP HQ camera. Image acquisition was controlled by NIS-Elements software (Nikon). Image background correction was done using the National Institute of Health ImageJ rolling-ball background subtraction plug-in (8 ). Cellular regions were determined using a watershed-based segmentation algorithm (9 (link)) which first retrieves nuclear regions using DNA staining then combines multiple cytosolic region markers to identify cellular boundaries. Images were visually inspected, and images with severe focus, staining, or cell-segmentation artifacts were discarded. We identified ~1,000 cellular regions per marker/well after applying automated cell segmentation to our image data.
Te2000 epifluorescence microscope
Manufactured by Nikon
Sourced in Japan
The Nikon TE2000 epifluorescence microscope is a laboratory equipment designed for fluorescence imaging applications. The core function of this microscope is to provide a platform for the visualization and analysis of fluorescently labeled samples.
Automatically generated - may contain errors
Lab products found in correlation
Nis elements software, by Nikon (2 mentions)
Cryostat, by Leica (2 mentions)
Custom filter set, by Chroma Technology (2 mentions)
Tissue tek optimum cutting temperature compound, by Sakura Finetek (1 mentions)
Monoclonal anti vinculin antibody, by Merck Group (1 mentions)
Antibody dilution buffer, by Roche (1 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Aniline blue, by Merck Group (1 mentions)
Amaxa system, by Lonza (1 mentions)
Px458 vector, by Addgene (1 mentions)
Partially complementary oligonucleotides, by Integrated DNA Technologies (1 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Coolsnap hq2 ccd camera, by Nikon (1 mentions)
Mastercycler ep gradient s thermocycler, by Eppendorf (1 mentions)
Nis elements ar 3, by Nikon (1 mentions)
Genome analyzer iix gaiix, by Illumina (1 mentions)
Ixon em du 897 emccd camera, by Oxford Instruments (1 mentions)
Volocity software, by PerkinElmer (1 mentions)
14 protocols using te2000 epifluorescence microscope
1
Fluorescence Imaging and Cell Segmentation
2
Hemocyte Membrane Potential Measurement
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Hemocytes were collected and washed as described above. Hemocyte counts were determined with a Neubauer hemocytometer, with three replicates per treatment. To measure the membrane potential of hemocytes, 104 hemocytes were seeded in a 96-well plate and given 15 min to attach. At that time, Bis-(1.3-Dibutylbarbituric Acid) Trimethine Oxonol (DiBAC4(3)) was added to 1 µg/mL and allowed to incubate for 10 min in the dark. Hemocytes were imaged and analyzed as previously described [21 (link)]. In brief, images were captured with a DS-Qi1Mc monochromatic camera on a Nikon TE2000 epifluorescence microscope with FITC filters. Cell intensity was collected by randomly choosing individual cells and manually generating the outline of cells as a region of interest (ROI). Normalized mean intensity (NMI) was calculated for each ROI by normalizing the ROI mean intensity (RMI) to background (BACK) and ambient light (AMB) by the calculation,
A minimum of 165 cells were analyzed across 3 replicates for each treatment.
A minimum of 165 cells were analyzed across 3 replicates for each treatment.
3
SARS-CoV-2 Spike Cell Fusion Assays
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For the transient transfection cell fusion assay producer 293T cells were co-transfected with pSV-TAT or pCMV-Tat and pcDNA-SARS-CoV-2-S, while target 293T cells were co-transfected with pLTR-LUC and pShuttle-hACE2 or hACE2 plasmids. At 48 h transfected cells were lifted, mixed 1:1, and after another 16–24 h cells were lysed and RLU measured by plate reader in 96-well format as described. Images of cell syncytia were captured with a Nikon TE2000 epifluorescence microscope running MetaMorph software. For the stable cell fusion assay HOS cell lines stably expressing HIV Tat and hACE2 (termed HOS-3734 and HOS-3742) were mixed 1:1 with TZMbl cells stably expressing S. After 16–24 h FFLUC activity was measured and syncytia images captured as described above. To observe LacZ activity, after cell fixation X-gal substrate was used as described. All experiments were performed with biological duplicates and repeated at least twice.
4
Tissue Distribution of NIR Fluorophore PH08 in Ovarian Cancer Mice
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To determine the tissue distribution of the NIR fluorophore, tumors and major organs were removed from ID8 ovarian cancer‐bearing mice 4 h post‐injection with 20 nmol of PH08 in 100 µL of 5% wt/v BSA/saline. The dissected tissues were trimmed and embedded in Tissue‐Tek optimum cutting temperature (OCT) compound (Sakura Finetek, Torrance, CA) without a pre‐fixation step, and the tissue block was frozen at −80 °C. Ten‐µm thick frozen sections were cut by a cryostat (Leica, Germany). The slides were subject to fluorescence analysis first, then stained for hematoxylin and eosin (H&E). Fluorescence and brightfield images were acquired on the 4‐channel Nikon TE2000 epifluorescence microscope. Image acquisition and analysis were performed using IPLab software (Scanalytics, Fairfax, VA). A custom filter set (Chroma Technology, Brattleboro, VT) composed of a 650/45 nm excitation filter, a 685 nm dichroic mirror, and a 720/60 nm emission filter was used for imaging. Exposure times were adjusted to obtain a similar maximum fluorescence value for each fluorescence image. Brightfield images of H&E‐stained slides from a matching field of view were also obtained.
5
Fluorescent Imaging of Cytoskeletal Structures
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Cells were stained for F-actin with Alexa Fluor® 555 tagged phalloidin (Life Technologies), for vinculin based focal adhesions with monoclonal anti-vinculin antibody (Sigma Aldrich), and for VE-cadherin based adherens junctions with VE-cadherin rabbit monoclonal antibody (Cell Signaling Technology). The nuclei were stained using ProLong® Gold antifade reagent with DAPI (Invitrogen). All antibodies were diluted using antibody dilution buffer (Ventana Medical Systems). Samples were fixed with 4% paraformaldehyde (Electron Microscopy Sciences, # 15714) for 10 minutes after rinsing twice with warm PBS, then permeabilized with 0.1% triton-x 100 (Sigma Aldrich) for 15 minutes. The sample was covered with a glass cover slip and sealed with nail polish. Images were captured using a Nikon TE-2000 epi-fluorescence microscope.
6
Quantifying Callose Deposition in Arabidopsis
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Cotyledons from 8-day-old B. rapa seedlings were treated with 1 µM flg22 or H2O; subsequently, they were treated with 95% ethanol, and stained with aniline blue as previously described (Gomez-Gomez et al., 1999 (link)) with minor modifications. Briefly, the cotyledons were incubated for at least 24 h in 95%–100% ethanol until the tissues were transparent. They were then washed with 0.07 M phosphate buffer (pH 9) and incubated for 1–2 h in 0.07 M phosphate buffer containing 0.01% (w/v) aniline blue (Sigma). At least ten cotyledons per condition per experiment were examined under ultraviolet light using a TE 2000 epifluorescence microscope (Nikon, Tokyo, Japan). The callose content was quantified using Photoshop CS6 software (Luna et al., 2011 (link)) to analyze digital photographs based on the number of white pixels (callose intensity) or the number of callose deposits relative to the total number of pixels covering the plant material.
7
Syk Protein Knockout in RBL-2H3 Cells
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Cas9-mediated DNA cleavage was used to knock out the endogenous gene coding for the Syk protein in RBL-2H3 cells via the insertion of a premature stop codon in the first exon of the gene. A highly specific single guide RNA (gRNA) (5′-GGCCAGAGCCGCAATTACCT-3′) targeting the first exon of rat Syk was designed using the http://crispr.mit.edu portal and then subcloned into PX458 vector (Addgene plasmid #48138) for simultaneous expression of the gRNA, WT Cas9, and a green fluorescent protein (GFP) reporter. For the gRNA subcloning, two partially complementary oligonucleotides (Integrated DNA Technologies) were assembled by PCR. Gel-purified PCR products were cloned into BbsI-digested PX458 using Gibson Assembly (NEB) following the manufacturer’s specifications. After cloning and sequencing, the final plasmid was used to transiently transfect RBL-2H3 cells using the Amaxa system (Lonza) following the manufacturer’s recommendations. Positive, GFP-expressing cells were selected by flow cytometry using an iCyt cell sorter and immediately plated at suboptimal concentration in 96-well plates. Subclones were screened using Western blotting to identify clones with no Syk expression. The absence of residual GFP expression in Syk KO clones was assessed using a Nikon TE2000 epifluorescence microscope.
8
Immunofluorescence Staining of Paraffin-Embedded Tissue
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Fixed brains were embedded in paraffin and tissue sections (5 μm) were used for staining. To remove excess paraffin, slides were immersed in xylene then rehydrated by incubation in 100, 95, and 75 % ethanol. Slides were washed in phosphate-buffered saline (PBS) then water. For antigen retrieval 10 mM citrate buffer (pH 6.0) was heated and slides were immersed for 20 minutes, followed by PBS washes. Sections were then permeabilized with 0.5 % Triton-X100 in PBS for 15 minutes, blocked in 5 % goat serum for 1 h, and incubated with primary antibodies overnight at 4 °C. Sections were washed three times in PBS and incubated with AlexaFluor-conjugated secondary antibodies (1:1000, Invitrogen) for 1 h at room temperature and counterstained with DAPI. Stained tissue sections were imaged using a Nikon TE2000 epifluorescence microscope.
9
Fluorescence Microscopy Imaging Protocol
10
Fluorescence Characterization of Calcium-Polymer Complexes
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5 mg/mL of various calcium salts were vortexed in a 5 μM aqueous solution of P800SO3-PEG (1 mL) for 30 min at room temperature. The mixture was centrifuged and washed twice with 1X PBS. A portion of the residual powder was soaked in 1X PBS (0.5 mL) and then added into a 24-well plate. A customized 4-channel Nikon TE2000 epifluorescence microscope was used to determine the fluorescence intensities of the dispersed samples. The fluorescence intensity of each cell was measured using ImageJ. All NIR fluorescence images were collected at identical exposure times and are displayed with equal normalization.
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