Paraffin-embedded sections from submandibular and lacrimal gland tissue from B6, SjSS, and MerKO mice (8 to 42 weeks of age) were deparaffinized, rehydrated, and unmasked by immersion in Trilogy (Cell Marque, Rocklin, CA, USA) solution, and heated in a pressure cooker for 15 min. Staining procedures for CD3+ T-cells and B220+ B-cells were followed, as described previously (1). Stained sections were visualized at 200× magnification using a Nikon Ti-E fluorescent microscope. Nikon NIS-Elements software (Nikon, Minato City, Tokyo, Japan) was used to determine the composition and size of infiltrates based upon the region of interest function (ROI), as previously described by our group (2).
Tie fluorescent microscope
Manufactured by Nikon
Sourced in United States
The TiE fluorescent microscope is a laboratory instrument designed for the observation and analysis of fluorescently labeled samples. It utilizes advanced fluorescence imaging technology to provide high-resolution, detailed images of biological specimens.
Automatically generated - may contain errors
Lab products found in correlation
Nis elements software, by Nikon (4 mentions)
35 mm glass bottom dishes, by MatTek (4 mentions)
Nis element, by Nikon (4 mentions)
Spectra x, by Lumencor (2 mentions)
Thioflavin s, by Merck Group (2 mentions)
Stage top incubator, by Tokai Hit (2 mentions)
Prolong gold mounting medium, by Thermo Fisher Scientific (2 mentions)
G3893, by Merck Group (2 mentions)
Goat anti mouse igg h l alexa fluor 568 conjugate, by Thermo Fisher Scientific (2 mentions)
Liperfluo, by Dojindo Laboratories (1 mentions)
Liperfluo, by Lumencor (1 mentions)
Ti e microscope, by Nikon (1 mentions)
V13241, by Thermo Fisher Scientific (1 mentions)
37 c stage top incubator, by Tokai Hit (1 mentions)
Mitosox red, by Thermo Fisher Scientific (1 mentions)
Mitosox red, by Lumencor (1 mentions)
Lsrfortessa, by BD (1 mentions)
Cm3050 cryostat, by Leica (1 mentions)
Dapi prolong gold anti fade reagent, by Thermo Fisher Scientific (1 mentions)
Primary cathepsin k, by Santa Cruz Biotechnology (1 mentions)
23 protocols using tie fluorescent microscope
1
Immunological Analysis of Salivary Gland
2
Scratch Wound Closure Assay
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SMCs were plated in 24 well plates at 50,000 cells per well in 2 mL of SBM media. After 16 h, 1 mL pipette tips were used to introduce a scratch in each well. SBM media was removed, and treatments comprised of 350 μL BM and 150 μL treatment (as defined in Section 2.5 ) were applied to wells in triplicate. The plate was transferred to a closed stage-top incubator (Tokai Hit Co., Bala Cynwyd, PA, USA) atop the motorized stage of an inverted Nikon TiE fluorescent microscope (Nikon, Inc., Melville, NY, USA) equipped with a 10×, 0.5 NA plan apochromatic lens (Nikon, Inc., Melville, NY, USA) and maintained at 37 °C and 5% CO2 for 24 h. Wells were imaged every hour for 48 h. Wound area was measured using ImageJ and wound closure is presented as the final would area relative to the initial wound area.
3
Calcium Regulation by Ryanodine Receptor 2 Modulation
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Cells were seeded on 35‐mm glass‐bottomed dishes and incubated with the calcium indicator, Fluo‐4 AM, for 20 minutes, and treated with 0.5 mmol/L of the ryanodine receptor 2 inhibitor, tetracaine hydrochloride, followed by peptide 7N3 (10 μmol/L). Images were acquired employing a closed, thermo‐controlled incubator atop the motorized stage of an inverted Nikon TiE fluorescent microscope (Nikon Instruments).
4
Live-cell Fluorescent Imaging of Cellular Processes
5
Thioflavin S Staining of Brain Sections
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The staining was performed as previously described [19 (link)]. Briefly, brain sections were deparaffinized and hydrated with the clearing agent xylene and a series of grade ethanol. After washed 3 times with PBS, the brain sections were incubated in filtered 1% Thioflavin S (Sigma-Aldrich, St. Louis, MO) for 8 minutes at room temperature. The tissues were washed twice in 70% ethanol, washed in PBS and mounted under a coverslip with anti-fading mounting media. The images were captured using the Nikon TiE fluorescent microscope (Nikon Instruments Inc., Melville, NY).
6
Cell Morphology and Migration Assays
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For cell morphology analyses and low-density migration assays, cells were seeded at 2.5 × 104 cells/well onto uncoated 24-well plates (Greiner Bio-One). For morphology, ×20 phase-contrast images were acquired on a Nikon TiE microscope (Nikon), and cell perimeters of individual cells were manually traced to define cell area using Fiji32 software. For low-density migration assays, cells were imaged every 2 minutes over a 2-hour period, using a ×20 objective on a Nikon TiE fluorescent microscope; then individual cells were tracked using the MTrackJ plugin of the Fiji32.
7
Live-cell Apoptosis Microscopy Assay
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MLE 12 cells cultured on 35-mm glass microwell dishes (MatTek) were imaged in a closed, thermo-controlled (37°C) incubator (Tokai Hit) atop the motorized stage of an inverted Nikon TiE fluorescent microscope (Nikon). Annexin V and propidium iodide dyes (V13241, ThermoFisher) were added with initiation of imaging. Dyes were excited using a diode-pumped light engine (SpectraX, Lumencor) and detected by ORCA-Flash 4.0 sCMOS camera (Hamamatsu Corporation) with Chroma Technology excitation/emission filters. Time-lapse large-area images (5 × 5 fields) were collected every 15 minutes using a 20× dry optic (0.75 numerical aperture) and stitched using NIS Elements (Nikon).
8
Quantifying Single-Walled Carbon Nanotubes in Cells
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Cells were seeded on 35 mm glass-bottom dishes (MatTek Corporation, Ashland, MA, USA) and incubated with the peroxynitrite indicator hydroxyphenyl fluorescein (HPF) (5 μM, Invitrogen, Eugene, OR, USA) for 15 min at 37 °C. Cells were washed with PBS, the media replaced, and the dish inserted in a closed, thermocontrolled (37 °C) stage top incubator (Tokai Hit Co., Shizuoka-ken, Japan) atop the motorized stage of an inverted Nikon TiE fluorescent microscope (Nikon Inc., Melville, NY, USA) equipped with a 60× oil immersion optic (Nikon, CFI PlanFluor, NA 1.43) and NIS Elements Software. HPF was excited using a Lumencor diode-pumped light engine (SpectraX, Lumencor Inc., Beaverton OR, USA) and detected using an FITC long pass filter set (Chroma Technology Corp, USA) and ORCA-Flash4.0 sCMOS camera (Hamamatsu Corporation, Bridgewater, NJ, USA). The number of SWCNTs per cell was quantified using the spots function in NIS Elements (Nikon Inc.).
9
Thioflavin S Staining of Brain Sections
10
Measuring Mitochondrial ROS in Alveolar Macrophages
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Cells were seeded on 35-mm glass bottom dishes (MatTek Corporation, Ashland, MA) and incubated with the superoxide indicator MitoSOX™ Red (5 μM, Invitrogen, Eugene, OR) for 15 min at 37 °C. Cells were washed with PBS, the media was replaced with exposure media, and the dish was inserted into a closed, thermo-controlled (37 °C) stage top incubator (Tokai Hit Co., Shizuoka-ken, Japan) atop the motorized stage of an inverted Nikon TiE fluorescent microscope (Nikon Inc., Melville, NY) equipped with a 60X oil immersion optic (Nikon, CFI PlanFluor, NA 1.43) and NIS Elements Software. MitoSOX™ Red was excited using a Lumencor diode-pumped light engine (SpectraX, Lumencor Inc., Beaverton OR) and detected using a DsRed longpass filter set (Chroma Technology Corp) and ORCA-Flash4.0 sCMOS camera (HAMAMATSU Corporation, Bridgewater, NJ). Data was collected on approximately 80 to 100 cells per stage position, with eight to ten stage positions in each of the separate experiments for 180 min. Data were analyzed using NIS Elements (Nikon Inc., Melville, NY). Data from three independent analyses for each particle size were used in the statistical calculations. Stage positions in which the particles did not result in alveolar macrophage (AM) generation of ROS were not used in the statistical analysis.
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