Neural tube explants were dissected from embryos cultured in 0.1 × MMR at 22°C to NF Stage (22 (link)-24 (link), (53 )) and plated onto poly-lysine (100μg/ml) and laminin-coated (20μg/ml) coverslips as described previously (60 (link)). Neuronal growth cones were imaged at room temperature 12-18 hours after plating. Live images were collected with a Yokogawa CSU5X1M 5000 spinning disk confocal on a Zeiss Axio Observer inverted motorized microscope with a Zeiss 63X Plan Apo 1.4 NA lens. Images were acquired with a Hamamatsu ORCA R2 CCD camera controlled with Zen software (Zeiss, Thornwood, MY). For time-lapse, images were collected every two seconds for one minute. Laser power for 561nm was 5-15%, with exposure time 600-1000ms. Microtubule dynamics were then quantified using plusTipTracker software (61 –63 (link)) with MATLAB version 2013a. The same parameters were used for all movies: maximum gap length: 8 frames; minimum track length: 3 frames; search radius range: 5 to 12 pixels; maximum forward angle: 50°; maximum backward angle: 10°; maximum shrinkage factor: 0.8; fluctuation radius: 2.5 pixels. Only cells with a minimum number of 10 track events in a movie were included for analysis.
Orca r2 ccd camera
Manufactured by Zeiss
Sourced in United States
The Orca R2 CCD camera is a high-performance scientific imaging device developed by Zeiss. It is designed to capture high-quality digital images for a variety of applications in scientific research and analysis. The camera features a large active area, high resolution, and low noise, making it suitable for a range of imaging tasks.
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Lab products found in correlation
Axio observer z1, by Zeiss (12 mentions)
Spinning disk confocal microscope, by Zeiss (5 mentions)
Zen 2012, by Zeiss (4 mentions)
Axio observer z1 microscope, by Zeiss (2 mentions)
Plan neofluar 40x 1.3 na, by Zeiss (2 mentions)
X 1.4 na, by Zeiss (2 mentions)
Plan neofluar, by Zeiss (2 mentions)
Axio observer, by Zeiss (1 mentions)
D8906, by Merck Group (1 mentions)
E coli trna, by Merck Group (1 mentions)
R4251, by Merck Group (1 mentions)
Am9342, by Thermo Fisher Scientific (1 mentions)
Am9763, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Arrayscan xti high content platform, by Thermo Fisher Scientific (1 mentions)
Fibronectin, by MatTek (1 mentions)
8 protocols using orca r2 ccd camera
1
Imaging Microtubule Dynamics in Neural Explants
2
Quantitative Analysis of MSC-Induced Target Cell Killing
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MSC-induced target cell killing was quantitatively analyzed by a spinning disk confocal microscope (Zeiss) equipped with a CSU-X1A 5000 spinning disk unit (Yokogowa Electric Corporation) multi-laser module with wavelengths of 458 nM, 488 nM, and 514 nM and an Axio Observer Z1 motorized inverted microscope equipped with a precision motorized XY stage (Carl Zeiss MicroImaging). Temperature was maintained at 37°C and 5% CO2 using an environmental control chamber. Zen 2012 software (Zeiss) was used to acquire images in time-lapse mode using a Zeiss Plan-Neofluar 20 × 0.4 NA objective on an Orca R2 CCD camera and to analyze average GFP and PI intensity. A quantitative digital image processing pipeline, created in ImageJ (W.S. Rasband, NIH [1997–2014]), was used to calculate specific PI incorporation of HUH7 and A549 cells. First, background intensities of PI channel were subtracted; then, raw images were thresholded based on their intensity histogram. The same threshold was used on all analyzed images. PI incorporation was determined as a multiplication of the mean pixel intensity and thresholded area.
3
Single Molecule RNA FISH in Neurons
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Neurons were plated on PDL-coated 28-mm glass bottom dishes (Invitrosci) and fixed 7 days later with 4% paraformaldehyde for 20 minutes at room temperature. Cells were permeabilized with cold 70% ethanol overnight at 4°C and washed the following morning for 5 minutes with wash buffer (25% formamide, 2X SSC). Neurons were stained with 40 20-nucleotide-long fluorescently labeled (Alexa-647) oligonucleotides designed against the Aag transcript overnight at 37°C in a heavily humidified chamber in hybridization buffer (100 mg/mL dextran sulfate, Sigma, D8906: 0.5 mg/mL E.coli tRNA, Sigma, R4251: 0.5 mg/mL ssDNA, Sigma D9156: 1 mg/mL Ultapure BSA, Ambion, AM2616: 10 mM VRC, New England Biolabs, S1402S: 25% formamide, Ambion, AM9342: 2X SSC, Ambion, AM9763). Finally, cells were stained with DAPI (2 μg/mL) in wash buffer for 30 minutes at 37°C before imaging. All the previous steps were done in nuclease-free solutions to avoid RNA degradation. We occasionally see faint transcripts in Aag-/- cells due to initiation of endogenous transcription before hitting the inserted cassette that disrupts the gene.
Images were taken with a Hamamatsu ORCA-R2 CCD Camera on a Zeiss Axio Observer.Z1 Microscope. 21 Z-stack images were taken, each 0.3 μm away from the previous. Images were compiled and foci per cell were counted with a MATLAB algorithm adapted from previously published methods [23 (link)].
Images were taken with a Hamamatsu ORCA-R2 CCD Camera on a Zeiss Axio Observer.Z1 Microscope. 21 Z-stack images were taken, each 0.3 μm away from the previous. Images were compiled and foci per cell were counted with a MATLAB algorithm adapted from previously published methods [23 (link)].
4
Spinning Disk Confocal Microscopy Imaging
5
Measuring Caspase-2 Proximity via BiFC
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Measurement of induced proximity of caspase 2 using the caspase-2 BiFC was performed as previously described42 (link). LN18 cells were plated in a 4-chamber dish containing coverslips (Nunc, Roskilde, Denmark) 24 h prior to transfection. Cells were transfected with caspase 2-CARD-VC (100 ng/well), caspase 2-CARD-VN (100 ng/well), and dsRed mito (20 ng/well) using lipofectamine 2000 (Life Technologies, Carlsbad, CA, USA). The following day, cells were treated with 290 nM MRZ or 15 nM BTZ. Cells were imaged using a spinning disk confocal microscope (Zeiss, Jena, Germany) equipped with a CSU-X1A 5000 spinning disk unit (Yokogowa Electric Corporation, Japan), multi-laser module with wavelengths of 458 nM, 488 nM, and 514 nM, and an Axio Observer Z1 motorized inverted microscope equipped with a precision motorized XY stage (Carl Zeiss MicroImaging, Thornwood, NY, USA). Temperature was maintained at 37 °C and 5% CO2 using an environmental control chamber. Zen 2012 software (Zeiss) was used to acquire images using a Zeiss Plan-Neofluar 40 × 1.3 NA objective on an Orca R2 CCD camera and to analyze average Venus intensity.
6
Immunofluorescence Staining of Cultured Neurons
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Neurons were plated in 24- or 96-well glass bottom plates (Invitrosci) and fixed with 4% paraformaldehyde for 15–30 minutes. Cells were then permeabilized with 0.1% Triton-X in CSK buffer (100 mM NaCl, 300 mM sucrose, 10 mM PIPES, 3 mM MgCl2, 1 mM EGTA) for 20 minutes, blocked with 4% bovine serum albumin (BSA) in PBS for 1 hour at 37°C, and stained overnight at 4°C with appropriate dilutions of antibodies in 4% BSA/PBS. Secondary antibodies were added for 2 hours and neurons were stained with Hoechst for 15 minutes. Images were taken with a Hamamatsu ORCA-R2 CCD Camera on a Zeiss Axio Observer.Z1 Microscope.
Neurons stained with anti-PAR were imaged and quantified with the ArrayScan XTI High Content Platform (Life Technologies). 7 images were taken per well at 10X magnification and the included software algorithm was used to quantify the total nuclear fluorescence using Hoechst staining as a nuclear mask.
Neurons stained with anti-PAR were imaged and quantified with the ArrayScan XTI High Content Platform (Life Technologies). 7 images were taken per well at 10X magnification and the included software algorithm was used to quantify the total nuclear fluorescence using Hoechst staining as a nuclear mask.
7
Spinning Disk Confocal Microscopy of Transfected Cells
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Cells were imaged using a spinning disk confocal microscope (Zeiss, Thornwood, NY, USA), equipped with a CSU-X1A 5000 spinning disk unit (Yokogowa Electric Corporation, Tokyo, Japan), multilaser module with wavelengths of 458, 488, 514 and 561 nm, and an Axio Observer Z1 motorized inverted microscope equipped with a precision motorized XY stage (Carl Zeiss MicroImaging, Thornwood, NY, USA). Images were acquired with a Zeiss Plan-Neofluar 40 × 1.3 NA or 64 × 1.4 NA objective on an Orca R2 CCD camera using Zen 2012 software (Zeiss, Thornwood, NY, USA). Cells were plated on dishes containing coverslips coated with fibronectin (Mattek Corp., Ashland, MA, USA) 24 h before transfection.
8
Spinning Disk Confocal Microscopy for Live-Cell Imaging
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Cells were imaged using a spinning disk confocal microscope (Zeiss, Thornwood, NY, USA), equipped with a CSU-X1A 5000 spinning disk unit (Yokogowa Electric Corporation, Japan), multi laser module with wavelengths of 458 nm, 488 nm, 514 nm, and 561 nm, and an Axio Observer Z1 motorized inverted microscope equipped with a precision motorized XY stage (Carl Zeiss MicroImaging, Thornwood, NY, USA). Images were acquired with a Zeiss Plan-Neofluar 40x 1.3 NA or 64x 1.4 NA objective on an Orca R2 CCD camera using Zen 2012 software (Zeiss, Thornwood, NY, USA). Cells were plated on dishes containing coverslips (Mattek Corp. Ashland, MA, USA) coated with poly-D-lysine hydrobromide 24 h prior to treatment. For time-lapse experiments, media on the cells was supplemented with Hepes (20 mM) and 2-mercaptoethanol (55 M). Cells were allowed to equilibrate to 37C in 5% CO2 prior to focusing on the cells.
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