All cells were fixed by incubation in 100% methanol for 10 minutes at -20°C. Fixed samples were blocked with blocking buffer [PBS (Corning), 1% Normal Donkey Serum (ThermoScientific), 1% BSA (Sigma Aldrich), 0.1% Triton X-100] for 1 hour at room temperature. Primary antibodies were diluted in blocking buffer and then incubated overnight at 4°C. Secondary antibodies were diluted in blocking buffer and then incubated 1 hour at room temperature in the dark. Antibodies are listed in Table 1 . Images were acquired using a Prime 95B camera mounted on a Nikon spinning disk microscope using a Plan Apo Lambda 20x objective lens. 9 randomized images per genotype and condition were captured. The software used for image acquisition and reconstruction were NIS-Elements Viewer (Nikon, Tokyo, Japan) and ImageJ (FIJI).
Prime 95b camera
Manufactured by Nikon
The Prime 95B is a high-performance camera designed for scientific and industrial applications. It features a 9.5-megapixel CMOS sensor and can capture images at up to 95 frames per second. The camera is capable of capturing detailed, high-resolution images and is suitable for a variety of applications that require accurate and consistent imaging.
Automatically generated - may contain errors
Lab products found in correlation
Nis elements viewer, by Nikon (2 mentions)
Spinning disk microscope, by Nikon (1 mentions)
Normal donkey serum (nds), by Thermo Fisher Scientific (1 mentions)
Plan apo lambda, by Nikon (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Corning (1 mentions)
Ti2 e microscope, by Nikon (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Matlab, by MathWorks (1 mentions)
Ti2 microscope, by Nikon (1 mentions)
Matlab, by National Instruments (1 mentions)
Live dead baclight fluorescent stain, by Thermo Fisher Scientific (1 mentions)
Eclipse te2000 microscope, by Nikon (1 mentions)
Alexa fluor secondary antibody, by Thermo Fisher Scientific (1 mentions)
Plan apo 60, by Nikon (1 mentions)
Ti2 e stand, by Nikon (1 mentions)
Eclipse ti2 e inverted microscope, by Nikon (1 mentions)
Orca flash 4.0 camera, by Hamamatsu Photonics (1 mentions)
Ix83 microscope, by Olympus (1 mentions)
Vectashield mounting medium, by Vector Laboratories (1 mentions)
11 protocols using prime 95b camera
1
Immunofluorescence Imaging of Fixed Cells
2
Quantification of DNA Damage in Canine Osteosarcoma Cells
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4.0 × 103 canine OS cells were plated in 96‐well collagen covered glass bottom plates and incubated overnight. For γH2A.X experiments, cells were incubated in media containing 0.1% DMSO, 1 μM doxorubicin for 2 hours, 1 μM verdinexor for 48 hours, or 1 μM doxorubicin for 2 hours followed by recovery in 1 μM verdinexor or 0.1% DMSO for 48 hours. Media was aspirated, cells were fixed with 4% paraformaldehyde and permeabilized with 1% Triton‐X. Cells were blocked in blocking buffer (1× PBS/2% BSA/0.1% Triton‐X) for 60 minutes and incubated with anti‐γH2A.X (Mouse mAb anti‐phospho‐Histone H2A.X [Ser 139] clone JBW30, Sigma‐Aldrich) at a dilution of 1:500 overnight at 4°C. Secondary donkey anti‐mouse antibody (Cat.#A‐21202, Alexa Fluor 488, Invitrogen) was applied for 1 hour. Cells were stained with DAPI for nuclear visualization and γH2A.X fluorescence was detected by immunofluorescence microscopy using a Nikon Ti2E microscope fitted with a Prime 95B camera. Data analysis was performed using custom MATLAB software (MathWorks, Natick, Massachusetts).33
3
Visualizing Fibroblast Vesicle Fusion Events
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The growth media for human fibroblasts were changed to DMEM FluoroBrite supplemented with 10% FBS 30 min before imaging. Cells were imaged using a TIRFM setup under a temperature-controlled environment (Tokai Hit) maintained at 37°C. Images were captured using Photometrics Prime 95B camera mounted on a Nikon Ti-2 microscope using Apo-TIRF 100×/1.49 oil-immersion or Apo-TIRF 60×/1.49 oil-immersion objective. Image acquisition was performed using NIS Elements Advanced Research Imaging software (version 4.60.00 [Build 1171] Patch 02). Images were processed and analyzed with NIS Elements and Matlab. Vesicle fusion events were analyzed by overlaying the time series images to determine all spots and by creating regions of interest around them as described previously (Ahmed et al., 2018 (link)). The fluorescence trace over time was extracted and analyzed using Matlab to determine bona-fide fusion events.
4
Assessing Plaque Acid Tolerance
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Plaque acid tolerance was evaluated using a previously validated method (Neilands et al., 2012 (link); Senneby et al., 2017 (link)). Briefly, 25 µl plaque sample was mixed with 75 µl TYE medium (1.7% tryptone, 0.3% yeast) containing 20 mM glucose and 40 mM phosphate/citrate buffer adjusted to pH 3.5 and incubated aerobically at 37°C for 2 hours. Following incubation, the cells were stained with LIVE/DEAD® BacLight™ Fluorescent Stain (Molecular Probes) and transferred into an Ibidi mini flow cells (Ibidi GmbH). Flow-cells were then viewed with confocal laser scanning microscopy (CLSM) using a Nikon Eclipse TE2000 microscope (Nikon Corp.) with an Ar laser (488 nm laser excitation). Images were acquired with a Photometrics Prime 95B camera using Nikon NIS-Elements software. Ten randomly selected images from each sample were saved for further analysis. All confocal images were examined by an experienced oral microbiologist and given a score 1-5 as described by Senneby et al. (2017) (link). This method has been shown to have high intra-rater agreement and the scoring corresponding well to the percentage obtained by manually counting the cells (Senneby et al., 2017 (link)).
5
Immunofluorescence Imaging of Neurospheres
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All neurospheres were fixed by incubation in 4% paraformaldehyde for 15 minutes at 4°C. Fixed samples were blocked with blocking buffer containing PBS/1% normal donkey serum/1% BSA/0.1% Triton X-100 for 1 hour at room temperature. Primary antibodies were diluted in blocking buffer and then incubated overnight at 4°C. Alexa Fluor secondary antibodies (Thermo Fisher Scientific) were diluted in blocking buffer and then incubated 1 hour at room temperature in the dark. Antibodies are listed in Table 2 . Hoechst (diluted 1:10,000 in 1X PBS) was applied to the slides for 20 minutes at room temperature. Images were acquired using a Prime 95B camera mounted on a Nikon spinning disk confocal microscope using a Plan Apo Lambda 20x objective lens at the Vanderbilt Nikon Center of Excellence. The software used for image acquisition and reconstruction were NIS-Elements Viewer (Nikon) and ImageJ (FIJI).
6
Integrin Distribution in FN Matrix
7
Live Imaging of Drosophila Embryogenesis
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Flies were raised at 25°C under standard conditions. Pupae were collected for imaging as described previously [52 ]. Ecad::GFP flies [53 (link)] were used for live imaging as previously described [1 (link)]. In brief, images were acquired with a spinning disk microscope from Gataca Systems driven by the MetaMorph software. The system is equipped with an inverted Nikon TI2E stand, a motorized XYZ stage, and a Nikon Plan Apo ×60 oil immersion (NA=1.4) lens and with a Prime95B camera.
8
Multimodal Live Cell Imaging
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Confocal images were acquired in a Nikon ECLIPSE Ti2-E inverted microscope with CSW-1 spinning disc system (Yokogawa), with a x100 CFI Plan Apo100x oil (na 1.45 wd 0.13 mm), equipped with temperature and CO2 control. Images were acquired using a Photometrics prime 95b camera and Nikon Elements software. Lasers 405/488/561/638 nm were used for DAPI/BFP, GFP, Cy3 and Cy5/MTDR, respectively. Live imaging time-lapse experiments were acquired in a wide field Olympus IX83 microscope (Olympus, Japan) equipped with ×100 oil immersion objective Plan Apo100x oil (na 1.45 wd 0.13 mm), Orca Flash 4.0 camera (Hamamatsu Photonics, Japan) and a LED light source (CoolLED, UK). Images were acquired using cellSense software and preprocessed using the online deblur tool.
9
Immunofluorescence Staining of Cellular Structures
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Cells for immunofluorescence were seeded on coverslips in 24-well plates and transfected with 0.5–0.75 µg plasmid using FuGENE6. 18 h after transfection, cells were fixed with different methods. For staining of astral MTs in Fig. 2 A and Fig. S2 B and for staining of cold-resistant spindle MTs in Fig. 2 L , cells were fixed in 4% formaldehyde and 0.1% glutaraldehyde in 37°C prewarmed MRB80 buffer for 10 min at room temperature. For staining of MTs in Fig. S4, E and F and Fig. S5, A and F , cells were sequentially fixed in −20°C methanol for 5 min and 4% formaldehyde in PBS for 5 min at room temperature. For staining of other proteins, cells were fixed with −20°C methanol for 10 min. Cell membranes were permeabilized with 0.15% Triton-X 100 in PBS, and subsequent blocking and labeling steps were performed in PBS supplemented with 2% BSA and 0.05% Tween-20. The permeabilizing, blocking, and labeling steps were performed at room temperature. Finally, coverslips were rinsed with 70% and 100% ethanol, air-dried, and mounted on glass slides with Vectashield mounting medium (Vector Laboratories). Slides were stored at −20°C.
Images were captured using Nikon Ni-U with 60× 1.40 NA oil objective equipped with DS-Qi2 camera (Nikon) for single-slice acquisition or Nikon Eclipse Ti2-E with 100× 1.45 NA oil objective equipped with Prime 95B camera for 3D acquisition with 0.11 µm z steps.
Images were captured using Nikon Ni-U with 60× 1.40 NA oil objective equipped with DS-Qi2 camera (Nikon) for single-slice acquisition or Nikon Eclipse Ti2-E with 100× 1.45 NA oil objective equipped with Prime 95B camera for 3D acquisition with 0.11 µm z steps.
10
GUS Assay and Imaging Protocol
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A GUS assay was performed on plants fixed in the 90% Acetone and cooled to -20℃. Stained plants were mounted in clearing media (Soukup, 2014) for observation.
OLYMPUS BX51 and Apogee U4000 camera, Nikon ECLIPSE 90i fit with Andor Zyla 5.5 w and Zeiss LSM880 confocal microscope and CSU-X1 Yokogawa spinning disk head and Photometrics Prime 95B camera fitted to a Nikon Ti-E inverted microscope were used for imaging. NIS-elements, Zeiss ZEN, and ImageJ (https://imagej.nih.gov/ij/) programs were used for image acquisition and processing. The image processing Images were acquired in 16bit depth and adjustments (brightness, Gamma 0,8) were applied to the entire image (Fig. 6 and7).
OLYMPUS BX51 and Apogee U4000 camera, Nikon ECLIPSE 90i fit with Andor Zyla 5.5 w and Zeiss LSM880 confocal microscope and CSU-X1 Yokogawa spinning disk head and Photometrics Prime 95B camera fitted to a Nikon Ti-E inverted microscope were used for imaging. NIS-elements, Zeiss ZEN, and ImageJ (https://imagej.nih.gov/ij/) programs were used for image acquisition and processing. The image processing Images were acquired in 16bit depth and adjustments (brightness, Gamma 0,8) were applied to the entire image (Fig. 6 and7).
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