T cell migration was evaluated by stacking a T cell containing Stacks plate on top of a plate containing 22Rv1 tumor cells in each well. T cells were allowed to migrate through the matrix for 24 h before the Stacks layers were separated. T cells were then fixed at their location within the collagen‐fibronectin matrix with a Foxp3 Fixation/Permeabilization kit (eBioscience, Thermo Fisher Scientific, Waltham, MA, USA), washed, and then stored at 4°C. Staining was performed by adding a solution of 20 mM Hoechst at a 1:250 dilution, anti‐CD4 (BD Biosciences, Franklin Lake, NJ, USA, #564419) at 1:50, and anti‐CD8 (BD Biosciences, USA #555367) at 1:100 to each well followed by overnight incubation at 4°C (reagents listed in Table S1 ). Each well was imaged on a Yokogawa CSU‐W1 Nikon Spinning Disk Confocal Microscope (Tokyo, Japan). Images were analyzed with NIS‐Elements software (RRID:SCR_014329; Nikon Instruments, Melville, NY, USA). CD4+ or CD8+ populations were identified by thresholding for signal intensities in the FITC and PE channels, respectively. Total migration distance for each cell population was quantified by calculating the distance that each cell had moved from the top of the well. Average migration was calculated by dividing the total migration distance by the total number of cells detected and correcting for the step size of the microscope during imaging.
Csu w1 spinning disk confocal microscope
Manufactured by Nikon
Sourced in Japan
The Nikon CSU-W1 is a spinning disk confocal microscope. It is designed for high-speed, high-resolution imaging of live cells and samples. The CSU-W1 utilizes a rotating disk with multiple pinholes to provide optical sectioning and improve image contrast.
Automatically generated - may contain errors
Lab products found in correlation
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (4 mentions)
Colcemid, by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Anti cd8, by BD (1 mentions)
Foxp3 fixation permeabilization kit, by Thermo Fisher Scientific (1 mentions)
Nis elements software, by Nikon (1 mentions)
Anti cd4, by BD (1 mentions)
Af568, by Thermo Fisher Scientific (1 mentions)
Imager z1 microscope, by Zeiss (1 mentions)
Hoechst dye, by Thermo Fisher Scientific (1 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
A11120, by Merck Group (1 mentions)
Ab152, by Merck Group (1 mentions)
Mab318, by Thermo Fisher Scientific (1 mentions)
Prolong diamond antifade mountant, by Thermo Fisher Scientific (1 mentions)
Nis element, by Nikon (1 mentions)
A 21235, by Thermo Fisher Scientific (1 mentions)
Infinity5 camera, by Teledyne (1 mentions)
Mabt868, by Merck Group (1 mentions)
Megascript t7 kit, by Thermo Fisher Scientific (1 mentions)
14 protocols using csu w1 spinning disk confocal microscope
1
Evaluating T Cell Migration
2
Quantitative Analysis of Microglia Morphology
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Hemi-brains with synaptosome injection were fixed in 4% PFA overnight, cryo-protected in 30% sucrose solution in 1 × PBS and sliced in 20-µm sections. Sliced tissues were stained with Iba1 (Fujifilm Wako Pure Chemical Corporation, 019–19741) followed by a secondary antibody staining (goat anti-rabbit AF568, Invitrogen, A-11011). DAPI was used for nuclear staining. Images close to the injection site (Additional file 1 : Fig. S1a) were taken using a Zeiss Imager Z1 microscope under a 20× objective lens. Tissues from bone marrow chimeras were processed and stained as described above. Images were taken using a CSU-W1 Nikon Spinning Disk Confocal microscope under 10× air, 20× air or 100× immerse oil lenses. All images were analyzed using the Fiji/ImageJ software by experimenters blinded to sample information.
3
Quantitative Telomere Fluorescence Analysis
4
Quantitative Telomere Fluorescence Analysis
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Q-FISH was performed as we previously described [28 (link)]. Briefly, all cells were maintained in complete medium, treated with 150 μM H2O2 for 1 hour or left untreated. Then, cells were incubated in medium containing 0.1 μg/ml colcemid (Millipore Sigma) for 4 hours to arrest the cells in metaphase. After adding hypotonic 0.075 M KCl buffer, the cells were fixed in cold methanol/acetic acid (3:1) and kept over-night at 4° C. Metaphase spreads were made and telomere FISH was performed by using Alexa546-conjugated Telomere DNA probe (TTAGGG)x3 (IDT). Chromosomes were counterstained with DAPI. For quantitative assessment of telomere length, digital images of chromosomes in metaphase and telomeres were captured by Nikon CSU-W1 Spinning Disk Confocal microscope, followed by quantitation of telomere size and visualization of telomere fluorescence intensity by using the Telometer plugin (available at http://demarzolab.pathology.jhmi.edu/telometer/index.html ) for FIJI software [29 (link)]. Additionally, the statical analyses of the average telomere intensities was performed by two-way ANOVAs followed by Fisher’s LSD separate post-hoc comparisons.
5
TWIST Protein Localization Assay
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Vero cells were seeded on coverslips and transfected with pTWIST-RuV-245-E2E1 and pTWIST-RuV-245-E2E1W448R expression constructs. 48 h post-transfection cells were fixed using 4% PFA, permeabilized with 0.2% TritonX-100, stained with mAb E1-18 and E2-1 (1:50 dilution), and the appropriate isotype-specific Alexa Fluor-conjugated secondary antibody (1:2500, Thermo Fisher) and Hoechst dye (1:10,000, Thermo Fisher). Images of 15–20 randomly chosen cells for each construct were acquired using a 100× oil objective on a Nikon CSU-W1 Spinning Disk confocal microscope at the Einstein Analytical Imaging Facility. Z-stacks were acquired for the entire depth of the cell at a step size of 0.2 µm. Figures were prepared using the ImageJ QuickFigures plugin (NIH) and Inkscape.
6
VLP Cellular Uptake Imaging Protocol
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Cells were cultured at a density
of 2.5 × 105 cells/well on glass-bottom Ibidi μ-Slide
8-well plates. After 24 h, the culture media was aspirated and the
cells were washed once with 1× PBS, then AF647-labeled VLPs in
OptiMEM were added (25 nM VLP final concentration). The cells were
incubated with particles in a humidified incubator (37 °C, 5%
CO2) for 120 min, then washed with 1× PBS (2×),
and stained with 10 μg/mL Hoescht 33342 in 1× PBS for 5
min. The cells were imaged immediately in OptiMEM on a Nikon CSU-W1
spinning disk confocal microscope.
of 2.5 × 105 cells/well on glass-bottom Ibidi μ-Slide
8-well plates. After 24 h, the culture media was aspirated and the
cells were washed once with 1× PBS, then AF647-labeled VLPs in
OptiMEM were added (25 nM VLP final concentration). The cells were
incubated with particles in a humidified incubator (37 °C, 5%
CO2) for 120 min, then washed with 1× PBS (2×),
and stained with 10 μg/mL Hoescht 33342 in 1× PBS for 5
min. The cells were imaged immediately in OptiMEM on a Nikon CSU-W1
spinning disk confocal microscope.
7
Quantitative Analysis of Myelination Dynamics
8
Immunostaining of Transgenic Mouse Brains
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Immunostaining of brain sections was used to validate AAV-directed gene targeting or expression (mice were euthanized 2 to 3 weeks after AAV administration) and to image DA neuron neurite density in the NAc. Brain tissues were collected after perfusion with PBS and 4% paraformaldehyde (PFA). PFA-fixed brain tissues were cryostat-sectioned at 40 μm thickness. Sections were permeabilized with 0.5% Triton X-100 for 30 min and then blocked in 5% normal goat serum for 1 hour at room temperature. Free-floating coronal sections were immunostained with anti-Becn2 (Millipore, ABC253), anti-GFP (Invitrogen, A11120), and anti-TH (Millipore, AB152 and MAB318) antibodies and then with Alexa Fluor or Alexa Fluor Plus 488–conjugated secondary antibody (Invitrogen, A-11008, A-11001, and A32723) and Alexa Fluor 594–conjugated secondary antibody (Invitrogen, A-11012 and A-11005). Slides were mounted using ProLong Diamond Antifade Mountant (Invitrogen, P36961). Fluorescence images were acquired using a Nikon CSU-W1 spinning disk confocal microscope. The images were analyzed using Nikon’s NIS-Elements.
9
Spatiotemporal Expression Analysis of Developmental Genes
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Fragments of chordin and Hox genes were isolated as previously described24 (link) using gene-specific oligonucleotides and a T7 adaptor. Riboprobes were synthesized using a T7 MEGAscript kit (ThermoFisher, AM1334) and stored at a concentration of 50 ng µl–1 in hybridization buffer at –20 °C. Whole-mount in situ hybridization in embryonic, larval and juvenile stages were conducted as described elsewhere24 (link),26 (link). Antibody staining in larval stages of O. fusiformis, Magelona spp. and C. teleta was carried out as previously described23 (link),115 (link) using the following antibodies: mouse anti-acetyl-α-tubulin antibody, clone 6-11B-1, 1:800 dilution (Sigma-Aldrich, MABT868, RRID: AB_2819178) and goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor 647, 1:800 dilution (Thermo Fisher Scientific, A-21235, RRID: AB_2535804). Differential interface contrast images of the colorimetric in situ were obtained using a Leica 560 DMRA2 upright microscope equipped with an Infinity5 camera (Lumenera). Fluorescently stained samples were scanned using a Nikon CSU-W1 spinning disk confocal microscope.
10
Quantitative Telomere Length Analysis
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Q-FISH was performed as we previously described [49 (link)]. Briefly, all cells were maintained in the indicated conditions. On the harvesting day, 0.1 µg/mL colcemid (Invitrogen) was added for 6 h. Cells were collected by Accutase, incubated with 0.075 M KCl, fixed in cold methanol/acetic acid (3:1) and stored at 4 °C. Metaphase spreads were prepared as previously described [50 (link),51 (link)], and telomere FISH was performed using a red fluorescence-conjugated Telomere PNA probe (AF546-OO-CCCTAACCCTAACCCTAA) (IDT). Chromosomes were stained with 0.5 μg/mL DAPI. For quantification and fluorescence intensity of telomere size, metaphase spreads and telomeres were captured by Nikon CSU-W1 Spinning Disk Confocal microscope and analyzed by TFL-TELO [28 (link)].
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