na oil objective, by Nikon - Product details

1

Imaging Muscle Puncta in Immobilized Worms

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Worms were immobilized on 10% agarose pads with 0.3 µl of 0.1 µm diameter polystyrene microspheres (Polysciences 00876–15, 2.5% w/v suspension). Body muscles just anterior to the vulva were imaged. Images were taken with a Nikon A1R confocal, using a 60 X/1.49 NA oil objective, with Nyquist sampling. Image volumes spanning the muscle surface were collected (~10 planes/volume, 0.15 μm between planes, and 0.06 μm /pixel). Maximum intensity projections for each volume were auto-thresholded, and puncta were identified as round fluorescent objects (area >0.1 μm²), using analysis of particles. Mean fluorescent intensity in each punctum was analyzed in the raw images. All image analysis was done using FIJI.

Gao L., Zhao J., Ardiel E., Hall Q., Nurrish S, & Kaplan J.M. (2022). Shank promotes action potential repolarization by recruiting BK channels to calcium microdomains. eLife, 11, e75140.

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2

Quantifying Synaptic Active Zone Proteins

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Worms were immobilized on 10% agarose pads with 3 μL of 0.1 μm diameter polystyrene microspheres (Polysciences 00876–15, 2.5% w/v suspension). The dorsal nerve cord just anterior to the vulva was imaged. Images were taken with a Nikon A1R confocal, using a 60X/1.49 NA oil objective. Image volumes spanning the dorsal nerve cord were collected (20–30 planes/volume, 0.4 μm between planes, and 0.14 mm/pixel). Maximum intensity projections for each volume were auto-thresholded, and puncta were identified as round fluorescent objects (area >0.1 μm²), using analysis of particles. Mean fluorescent intensity in each punctum was analyzed in the raw images. Pre-synaptic regions of interest (ROIs) were identified by localization of an mCherry tagged synaptic vesicle marker (UNC-57/Endophilin) expressed in either the ACh or GABA motor neurons. Using CRISPR tagged alleles (see key resources table), the intensity of endogenously expressed active zone markers in the UNC-57 ROIs were quantified. All image analysis was done using FIJI.

Zhao J., Gao L., Nurrish S, & Kaplan J.M. (2023). Post-synaptic GABAA receptors potentiate transmission by recruiting CaV2 channels to their inputs. Cell reports, 42(10), 113161.

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3

Monitoring mast cell ROS and death

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To monitor and localize ROS production by live confocal imaging, mast cells (1.5 × 10⁶ cells) were immobilized overnight in MatTek glass bottom microwell dishes (MatTek Co., Ashland, MA) using a biocompatible anchor for membrane (BAM; SUNBRIGHT® OE-040CS, NOF Corporation, Tokyo, Japan). To label granules and detect ROS, cells were incubated with a cocktail of probes (50 nM LysoTracker^TM Red DND-99 and 5 μM CellROX^TM Deep Red) for 30 min. Cells were subsequently washed and kept in PBS. Images were immediately recorded at the indicated time intervals at 37 °C and 5% CO₂ at baseline (initial 72 min), and after addition of mefloquine (for another 112 min) using a Nikon Ti2-E microscope, equipped with an X-LIGHT V2 L-FOV spinning disk with a pinhole size of 60 µm (Crest Optics). A 100X/1.45 NA oil objective (Nikon) and a Prime 95B 25 mm camera (Photometrics) were used to capture the images. To monitor cell death by live confocal imaging, BAM-anchored WT and serglycin^−/− mast cells (1.5 × 10⁶ cells) were incubated with a cocktail of Annexin V and DRAQ7 in a glass bottom 6-well plate (MatTek Co.). Thereafter, cells were treated with either PBS or mefloquine and images were immediately recorded at indicated time intervals, at 37 °C and 5% CO₂, through a 40X/0.6 NA air objective on a Nikon Ti2-E microscope.

Paivandy A., Eriksson J., Melo F.R., Sellin M.E, & Pejler G. (2019). Lysosomotropic challenge of mast cells causes intra-granular reactive oxygen species production. Cell Death Discovery, 5, 95.

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4

Droplet Formation Assay for Protein Interactions

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Proteins dissolved in a buffer containing 20 mM HEPES, pH 7.4, 500 mM NaCl were mixed, and the concentration of NaCl was adjusted to 150 mM with a buffer containing 20 mM HEPES, pH 7.4. The mixture was treated immediately with PEG8000 or PreScission protease (a gift from Dr. Hong Zhang, Institute of Biophysics, Chinese Academy of Sciences), and the concentration of NaCl was further adjusted to 150 mM NaCl and then droplet formation was examined. For imaging, droplets were observed either on a glass slide or in a glass-bottom cell culture dish for differential interference contrast (DIC) or fluorescence imaging. Imaging was with a NIKON A1 HD25 microscope equipped with a 100X/1.45 NA oil objective (1,024 × 1,024 pixels) at room temperature. NIS-Elements AR analysis was used to analyze these images. The FWHM (Full width at half maximum) of the droplets was measured to define the thresholding values in algorithms for subsequent size measurement by NIS-Elements AR Analysis.

Kang K., Shi Q., Wang X, & Chen Y.G. (2022). Dishevelled phase separation promotes Wnt signalosome assembly and destruction complex disassembly. The Journal of Cell Biology, 221(12), e202205069.

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5

Quantification of Muscarinic Receptor Activation

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In some experiments, the fluorescence signals of HEK293T cells transfected with the muscarinic receptor-based chimeric
constructs were measured with a TECAN Safire2 fluorescence plate reader (TECAN, Männedorf, Switzerland; excitation, 480 nm;
emission, 520 nm). During the measurement, the culture media was replaced with 100 μl Tyrode solution containing ACh at
varied concentrations from 0–100 μM. The ΔF/F₀ of each construct was obtained by averaging the
ACh-induced fluorescence responses of transfected wells after digitally subtracting that of neighboring control non-transfected
wells.
In other culture cell experiments, HEK293T cells and cultured neurons were imaged by an inverted Nikon Ti-E A1 confocal
microscope with a 40×/1.35 NA oil objective (Nikon, Tokyo, Japan). Cells were perfused with standard extracellular Tyrode
solution containing (in mM): 150 NaCl, 4 KCl, 2 MgCl₂, 2 CaCl₂, 10 HEPES and 10 Glucose, with pH of 7.4, in
an imaging chamber during imaging. Agonist acetylcholine (Solarbio, Beijing, China), tiotropium bromide (Dexinjia Bio & Tech
Co., Ltd, Jinan, China), and AF-DX 384 (Sigma-Aldrich) were delivered with a custom-made perfusion system and/or bath applied. The
chamber was washed with Tyrode solution between applications and cleaned with 75% ethanol between experiments.

Jing M., Zhang P., Wang G., Feng J., Mesik L., Zeng J., Jiang H., Wang S., Looby J.C., Guagliardo N.A., Langma L.W., Lu J., Zuo Y., Talmage D.A., Role L.W., Barrett P.Q., Zhang L.I., Luo M., Song Y., Zhu J.J, & Li Y. (2018). ED SUM: Signaling by the neurotransmitter acetylcholine is monitored in cells and animals with a sensitive reporter.: A genetically encoded fluorescent acetylcholine indicator for in vitro and in vivo studies. Nature biotechnology, 36(8), 726-737.

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6

Quantification of Muscarinic Receptor Activation

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In some experiments, the fluorescence signals of HEK293T cells transfected with the muscarinic receptor-based chimeric
constructs were measured with a TECAN Safire2 fluorescence plate reader (TECAN, Männedorf, Switzerland; excitation, 480 nm;
emission, 520 nm). During the measurement, the culture media was replaced with 100 μl Tyrode solution containing ACh at
varied concentrations from 0–100 μM. The ΔF/F₀ of each construct was obtained by averaging the
ACh-induced fluorescence responses of transfected wells after digitally subtracting that of neighboring control non-transfected
wells.
In other culture cell experiments, HEK293T cells and cultured neurons were imaged by an inverted Nikon Ti-E A1 confocal
microscope with a 40×/1.35 NA oil objective (Nikon, Tokyo, Japan). Cells were perfused with standard extracellular Tyrode
solution containing (in mM): 150 NaCl, 4 KCl, 2 MgCl₂, 2 CaCl₂, 10 HEPES and 10 Glucose, with pH of 7.4, in
an imaging chamber during imaging. Agonist acetylcholine (Solarbio, Beijing, China), tiotropium bromide (Dexinjia Bio & Tech
Co., Ltd, Jinan, China), and AF-DX 384 (Sigma-Aldrich) were delivered with a custom-made perfusion system and/or bath applied. The
chamber was washed with Tyrode solution between applications and cleaned with 75% ethanol between experiments.

Jing M., Zhang P., Wang G., Feng J., Mesik L., Zeng J., Jiang H., Wang S., Looby J.C., Guagliardo N.A., Langma L.W., Lu J., Zuo Y., Talmage D.A., Role L.W., Barrett P.Q., Zhang L.I., Luo M., Song Y., Zhu J.J, & Li Y. (2018). ED SUM: Signaling by the neurotransmitter acetylcholine is monitored in cells and animals with a sensitive reporter.: A genetically encoded fluorescent acetylcholine indicator for in vitro and in vivo studies. Nature biotechnology, 36(8), 726-737.

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7

Visualizing grk mRNA in Drosophila Oocytes

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smFISH was performed on ovaries dissected from well-fed females as described previously (Abbaszadeh and Gavis, 2016 (link)) using probes for the coding region of grk conjugated to ATTO-565 (ATTO-Tec). DNA was visualized with DAPI (1:1000; Molecular Probes). Oocytes were imaged using a Nikon A1R confocal microscope with a 60x/1.4 NA oil objective.

Tamayo J.V., Teramoto T., Chatterjee S., Tanaka Hall T.M, & Gavis E.R. (2017). The Drosophila hnRNP F/H Homolog Glorund Uses Two Distinct RNA-binding Modes to Diversify Target Recognition. Cell reports, 19(1), 150-161.

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8

FRAP Analysis of Tagged Proteins in Candida-Infected Cells

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FRAP experiments of GFP or RFP-tagged proteins, transiently expressed in RAW-Dectin1 cells, were conducted on an A1R point-scanning confocal system (Nikon Instruments, Japan). For FRAP, Candida-BFP hyphae-infected cells were imaged in HPMI at 37°C. Images were acquired using a 60x/1.4 NA oil objective (Nikon), 1.2-AU pinhole, resonant scanning mode, and 16x line averaging. For a complete 2 min FRAP acquisition at 1.9 fps, after 5 s of initial imaging, a region of interest 3 μm in diameter was bleached for 1.06 s using the 405 laser at 100% power, followed by imaging for fluorescence recovery. Images were exported and analyzed for fluorescence intensity using Volocity software.
After background subtraction, fluorescence intensity units were normalized (see Figure 6 legend) using Microsoft Excel software, and transformed to a 0–1 scale, to correct for differences in bleaching depth and allow for comparison of up to 30 individual FRAP curves per condition. Graphpad Prism software was used to fit the FRAP curves to a single exponential, plotted as fractional recovery over time.

Maxson M.E., Naj X., O'Meara T.R., Plumb J.D., Cowen L.E, & Grinstein S. (2018). Integrin-based diffusion barrier separates membrane domains enabling the formation of microbiostatic frustrated phagosomes. eLife, 7, e34798.

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Na oil objective

Lab products found in correlation

8 protocols using na oil objective

Imaging Muscle Puncta in Immobilized Worms

Quantifying Synaptic Active Zone Proteins

Monitoring mast cell ROS and death

Droplet Formation Assay for Protein Interactions

Quantification of Muscarinic Receptor Activation

Quantification of Muscarinic Receptor Activation

Visualizing grk mRNA in Drosophila Oocytes

FRAP Analysis of Tagged Proteins in Candida-Infected Cells

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