Confocal microscope

Manufactured by Nikon

Sourced in Japan, United States, Germany, China, Switzerland, France

The Nikon Confocal Microscope is an advanced imaging instrument designed to capture high-resolution, three-dimensional images of samples. It uses a focused laser beam to scan the specimen and detect the reflected or emitted light, allowing for the creation of detailed, optical sections of the sample.

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Nikon Eclipse Ti-U confocal microscope Ti-E confocal microscope A1R-Ti2 confocal microscope Nikon A1 confocal/Eclipse Ti inverted microscope system LSM 800 confocal microscope D-Eclipse C1 confocal microscope Nikon CE1 Confocal Microscope A1 MP confocal microscope LSM 700 confocal microscope C2 Silaser-scanning confocal microscope

523 protocols using confocal microscope

Visualizing Mitotic Progression in HeLa Cells

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HeLa-Teton cells stably expressing H2B-mCherry were treated as indicated (Figure 4), and then long-term imaging was performed. Images were collected every 6 min for 12–15 h using a Nikon confocal microscope equipped with an environment chamber that controls temperature and CO₂, and 20× objective. Image panels displaying the elapsed time between consecutive frames were assembled using the software designed for Nikon confocal microscope. The time taken for each cell to progress from NEB to anaphase onset (chromatid separation) was calculated in minutes and plotted in GraphPad Prism. The experiments in Figure 4 were repeated at least two times and the results were highly reproducible. Quantification was performed based on the results from a single experiment. Average and SD were calculated using GraphPad Prism.
For live-cell imaging in Figure 2, E and G, GFP-Ska3 WT, 2A, 4A-3, 6A, and 6D were expressed in HeLa Tet-On cells. After being treated with MG132 for 1 h, cells were immediately subjected to imaging using a Nikon confocal microscope. Images were processed in ImageJ and Adobe Photoshop.

Zhang Q., Hu L., Chen Y., Tian W, & Liu H. (2020). Multisite phosphorylation determines the formation of Ska–Ndc80 macro-complexes that are essential for chromosome segregation during mitosis. Molecular Biology of the Cell, 31(17), 1892-1903.

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Dye-based Neurotransmitter Release Assay

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Fluorescent dye FM1-43 was used to mimic the release of neurotransmitters as it can be incorporated into the synaptic vesicle membrane, and the release of neurotransmitters was measured by the decrease of FM1-43 fluorescence intensity in primary neurons. Firstly, DMEM medium containing MPP⁺ (50 μM) and APROM₂ (20 μg/mL) or CeNPs (20 μg/mL) was added into culture dishes, and primary neurons were cultured for 24 h. Then, primary neurons were incubated with HEPES buffer containing 100 μM FM1-43, 1 mM Ca²⁺ and 30 mM K⁺ for 2 min, and the fluorescent signal was tested by using a Nikon confocal microscope (Nikon, Japan). Subsequently, neurons were incubated with HEPES buffer containing 1 mM Ca²⁺ and 15 mM K⁺ for 5 min, during which FM1-43 contained synaptic vesicles were released. The fluorescent signal was detected again by using a Nikon confocal microscope.

Lin P., Zhang B., Yang H., Yang S., Xue P., Chen Y., Yu S., Zhang J., Zhang Y., Chen L., Fan C., Li F, & Ling D. (2024). An artificial protein modulator reprogramming neuronal protein functions. Nature Communications, 15, 2039.

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Multifaceted Neuronal Profiling in Rat PrL

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Rats were perfused through the ascending aorta with 4% paraformaldehyde under anesthesia. The PrL tissues were cut into 25 µm-thick transverse sections after 30% DEPC–sucrose dehydration at 4 ℃ and hybridized at 42 ℃ for 16 h with the 5ʹ-TYE665-label Acp5 probe 5ʹ-ACGTATCCATCACCAATCTCT-3ʹ (1:200, QIAGEN). The sections were then incubated at 4℃ overnight with primary antibodies against NeuN (Millipore, 1:500), Iba1 (Abcam, 1:400), GFAP (CST, 1:400), CaMKIIa (Abcam, 1:200), PV (Novusbio, 1:400) or SST (ABclonal, 1:100). After that, the sections were incubated with fluorescein isothiocyanate-conjugated secondary antibody at 37 °C for 60 min. The stained sections were examined using with a Nikon confocal microscope equipped.
For IL-6R immunofluorescence, the PrL tissues were cut into 25 µm-thick transverse sections and incubated with primary antibodies against IL-6R (Santa, 1:50), NeuN (Millipore, 1:500), Iba1 (Abcam, 1:400) or GFAP (Abcam, 1:400) at 4 ℃ overnight. After that, the sections were incubated with Cy3 or fluorescein isothiocyanate-conjugated secondary antibody at 37 °C for 60 min. The stained sections were examined using with a Nikon confocal microscope equipped, and images were captured with a Nikon DS-Qi2 camera.

Zhao Y.T., Deng J., Liu H.M., Wei J.Y., Fan H.T., Liu M., Xu T., Chen T.F., He J.Y., Sun W.M., Jia T.Y., Zhang X.Q, & Xin W.J. (2022). Adaptation of prelimbic cortex mediated by IL-6/STAT3/Acp5 pathway contributes to the comorbidity of neuropathic pain and depression in rats. Journal of Neuroinflammation, 19, 144.

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In Vitro Phase Separation Assay

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In vitro phase separation assay was performed in the phase separation buffer (20 mm Tris‐HCl, pH 7.5, 100 mm NaCl, 5 mm KH₂PO₄, 1.5 mm MgCl₂, and 1 mg mL⁻¹ BSA). Purified GFP‐TopBP1‐BRCT4‐5 and pre‐rRNA were added to the buffer with indicated concentrations, and PEG8000 (NEB) (final concentration, 5% (w/v)) was also added. The mixture was directly pipetted onto glass‐bottomed dishes (NEST), and observed by a Nikon confocal microscope with 40× differential interference contrast (DIC). The method used for pre‐rRNAs isolated from 293T cells has been described previously.^[⁵⁵^]For in vitro FRAP experiments, liquid droplets photobleached with 80% laser power for 1 s using 488‐nm lasers were performed on a Nikon confocal microscope with 40× differential interference contrast (DIC).

Xin D., Gai X., Ma Y., Li Z., Li Q, & Yu X. (2023). Pre‐rRNA Facilitates TopBP1‐Mediated DNA Double‐Strand Break Response. Advanced Science, 10(28), 2206931.

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Intracellular ROS Measurement in HUVECs

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The DCFH-DA, an oxidation-sensitive fluorescent probe was employed to suspend pure HUVECs and HUVECs were incubated in vitro for 20 minutes at the typical 37 °C. Using a confocal microscope (Nikon, Japan), cell fluorescence was captured after sufficient washing with serum-free media. Intracellular ROS was measured with the ROS assay kit (Beyotime, S0033) in accordance with the manufacturer's instructions.
ROS generation was calculated in vivo using DHE staining. Fresh skeletal muscle samples were cut into cryosections and treated with DHE (5 mol/L, Sigma Aldrich, D7008) for 30 minutes at 37 °C. The samples were then analysed using a confocal microscope (made by Nikon, Japan).

Chen F., Zhan J., Liu M., Mamun A.A., Huang S., Tao Y., Zhao J., Zhang Y., Xu Y., He Z., Du S., Lu W., Li X., Chen Z, & Xiao J. (2023). FGF2 Alleviates Microvascular Ischemia-Reperfusion Injury by KLF2-mediated Ferroptosis Inhibition and Antioxidant Responses. International Journal of Biological Sciences, 19(13), 4340-4359.

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Adipocyte Morphometry and Apoptosis Analysis

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The diameter and number of adipocytes were determined in 3-4 paraffin-embedded sections of the SAT. The sections were stained with H and E. After staining, the diameters of adipocytes were determined in three sections from young (n = 100 adipocytes) and old rats (n = 100 adipocytes), using the NDP. VIEW image software of NanoZoomer S60(HAMAMATSU, Japan). The number of adipocytes in the same area was counted from three sections from the two groups of rats (n = 3) using the NDP. VIEW image software.
The TUNEL assay was performed in each group (n = 3) using the Apoptotic Detection kit (Cat# MK500, Takara, Japan). All stained sections were imaged under a confocal microscope (Nikon, Japan).
The immunofluorescence assay was performed as previously described (Jeong et al., 2020 (link)). Briefly, sections (n = 3) were incubated in primary antibodies Cleaved Caspase-3 (Cat#9664, CST, United States) at 4 °C overnight and with a secondary antibody for 1 h at room temperature. All stained sections were imaged under a confocal microscope (Nikon, Japan).

Song X.H., He N., Xing Y.T., Jin X.Q., Li Y.W., Liu S.S., Gao Z.Y., Guo C., Wang J.J., Huang Y.Y., Hu H, & Wang L.L. (2021). A Novel Age-Related Circular RNA Circ-ATXN2 Inhibits Proliferation, Promotes Cell Death and Adipogenesis in Rat Adipose Tissue-Derived Stromal Cells. Frontiers in Genetics, 12, 761926.

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Measuring Intracellular and Mitochondrial ROS

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ROS in the total intracellular and TA muscle were examined using DCFH-DA or MitoSox Red staining (Ye Sen, Shanghai, China). For intracellular and mitochondrial ROS, the indicated cells were incubated with 10 μM DCFH-DA or 5 μM MitoSox Red at 37°C for 30 min, washed three times with PBS and analyzed using flow cytometry. To visualize mitochondrial ROS, the indicated cells were incubated with 100 nM MitoTracker Green FM and 5 μM MitoSox Red at 37°C for 30 min (Ye Sen). The cells were then washed three times with PBS and fixed with PFA for 20 min. Then, they were permeabilized with 0.15% Triton X-100 for 12 min and blocked with 10% FBS for 30 min. Lastly, the cells were stained with DAPI for 10 min and imaged with a confocal microscope (Nikon). For TA muscle ROS, the tissue was minced, and digested with 0.2% collagenase II and 0.05% trypsin for 40 min to obtain single-cell suspension. The cells were resuspended with 10 μM DCFH-DA in PBS for 30 min after the centrifugation, followed by washing three times with PBS and analyzed using flow cytometry. For the DCFH-DA staining of TA muscle, sections were washed with PBS and incubated with DCFH-DA (10 μM) at 37°C for 30 min. Then, sections were washed with PBS and incubated with DAPI at room temperature for 10 min to visualize nuclei. The stained sections were observed with a confocal microscope (Nikon).

Liu X., Zu E., Chang X., Ma X., Wang Z., Song X., Li X., Yu Q., Kamei K.I., Hayashi T., Mizuno K., Hattori S., Fujisaki H., Ikejima T, & Wang D.O. (2021). Bi-phasic effect of gelatin in myogenesis and skeletal muscle regeneration. Disease Models & Mechanisms, 14(12), dmm049290.

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Mitochondria-Lysosome Colocalization Analysis

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Briefly, the medium of cultured cells was discarded, and the cells were washed with phosphate buffer saline (PBS) 3 times. Then, the cells were fixed with fixation buffer (Biolegend) for 20 min, washed with PBS, and then incubated with membrane-rupturing reagents for another 20 min. Subsequently, after being washed with PBS, the prepared cells were coincubated with specific primary antibodies for 1 h. Then, after being counterstained with 4',6-diamidino-2-phenylindole (DAPI) for approximately 10 min, cell samples were observed and imaged with a confocal microscope (Nikon) or directly detected by flow cytometry analysis without nuclear staining. To assess mitochondrial colocalization with lysosomes, mitochondria from 293T cells expressing COX8A N-terminal signal peptide-mCherry fusion protein were isolated and incubated with a culture of normal 293T cells for 24 h, after which the cells were washed twice with PBS. Next, the cells were incubated with Lyso Dye (Cat. No. MD01, Dojindo Laboratories, Kumamoto, Japan) at 37 for 30 min. Then, after being counterstained with Hoechst 33342 at 37 for 10 min, the cells were observed and imaged with a confocal microscope (Nikon). All experimental procedures were conducted in darkness at room temperature (RT).

Xie Q., Zeng J.K., Xu F., Zheng Y., Li T., Ren J., Chen K., Zhang Q., Xie R., & Zhu J. (2020). Mitochondrial Transplantation Improves Stroke-Induced Brain Injury: Possible Involvement of Selective Component Recombination.

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Confocal Analysis of ADCP-mediated Cell Killing

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For confocal analysis ADCP assay was conducted in the same conditions as described above but adapted for later microscopy analysis. Briefly, 3T3-LASV GPC target cells were seeded in glass cover-slips and incubated with the respective sera conditions, and then J774A.1 macrophages previously stained with 1:1000 CellTrace® Far Red (see above) were added at a 1:1 Target to effector cell ratio to allow easy visualization. After 4 h, coverslips were washed and mounted in slides with DAPI containing mounting media (VECTASHIELD) and allowed to solidify O/N. Next, day samples were analyzed in a Nikon confocal microscope and further compiled through ImageJ software.

Abreu-Mota T., Hagen K.R., Cooper K., Jahrling P.B., Tan G., Wirblich C., Johnson R.F, & Schnell M.J. (2018). Non-neutralizing antibodies elicited by recombinant Lassa–Rabies vaccine are critical for protection against Lassa fever. Nature Communications, 9, 4223.

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Quantification of Perivascular Deposits

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Quantification was performed from maximum projections of 10-µm-thick Z-stack images taken on a BZ9000 fluorescent microscope subjected to threshold processing (Otsu) using ImageJ, as described previously (Montagne et al., 2018 (link)). Briefly, the amount of perivascular deposits of fibrin(ogen) and IgG was determined as integrated density of the deposits on the abluminal side of the lectin-positive vessels. Representative images were taken on a Nikon confocal microscope with standardized gain, digital offset, and laser intensity, and these conditions were kept identical for both groups. In each animal, five randomly selected fields from the cortex and hippocampus were analyzed in four nonadjacent sections (∼100 µm apart). Seven animals per group were analyzed.

Lazic D., Sagare A.P., Nikolakopoulou A.M., Griffin J.H., Vassar R, & Zlokovic B.V. (2019). 3K3A-activated protein C blocks amyloidogenic BACE1 pathway and improves functional outcome in mice. The Journal of Experimental Medicine, 216(2), 279-293.

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