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Ti confocal microscope

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The Nikon Ti confocal microscope is a high-performance imaging system designed for advanced microscopy applications. It features a modular design that allows for customization to meet specific research needs. The Ti confocal microscope provides superior optical performance, enabling high-resolution, multi-dimensional imaging of biological samples.

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Lab products found in correlation

Mitotracker green, by Thermo Fisher Scientific (2 mentions) Icc50 microscope, by Leica camera (1 mentions) Lysotracker deep red, by Thermo Fisher Scientific (1 mentions) H3570, by Thermo Fisher Scientific (1 mentions) M7514, by Thermo Fisher Scientific (1 mentions) L7528, by Thermo Fisher Scientific (1 mentions) Hoechst, by Thermo Fisher Scientific (1 mentions) Prolong gold mountant, by Thermo Fisher Scientific (1 mentions) Deadend fluorometric tunel system, by Promega (1 mentions) Ixon ultra 897 emccd camera, by Oxford Instruments (1 mentions) Nis element imaging software, by Nikon (1 mentions) Lysotracker dnd 99, by Thermo Fisher Scientific (1 mentions) Nucblue live cell nuclear stain, by Thermo Fisher Scientific (1 mentions) Opti mem, by Thermo Fisher Scientific (1 mentions)

20 protocols using ti confocal microscope

1

Doxorubicin Delivery Assay in Mice

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For the doxorubicin delivery assay, a minimum of five mice from saline and Minnelide were included for analysis. Mice received an intravenous infusion of doxorubicin 24 h after the final dose of Minnelide. Mice were euthanized and tissues processed as above. Sections were deparaffinised, rehydrated and counterstained with DAPI. Doxorubicin fluorescence was visualized under a Nikon Ti confocal microscope using standardized settings, and background signal intensities were established against samples without doxorubicin stain.
Banerjee S., Modi S., McGinn O., Zhao X., Dudeja V., Ramakrishnan S, & Saluja A.K. (2015). Impaired synthesis of stromal components in response to Minnelide improves vascular function, drug delivery and survival in pancreatic cancer. Clinical cancer research : an official journal of the American Association for Cancer Research, 22(2), 415-425.
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2

Imaging Tumor Sections with Confocal Microscopy

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To provide a high-resolution example image of a QCC, section 17 from control tumor 3 was imaged using a Nikon Ti confocal microscope at × 60 magnification (Fig. 2a). Merged and single-color images were labelled using Image J [23 (link)].
Kabraji S., Solé X., Huang Y., Bango C., Bowden M., Bardia A., Sgroi D., Loda M, & Ramaswamy S. (2017). AKT1low quiescent cancer cells persist after neoadjuvant chemotherapy in triple negative breast cancer. Breast Cancer Research : BCR, 19, 88.
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3

Immunostaining of S. pombe for Swi6 Localization

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Cells were grown to mid log phase, OD 600nm =0.2–0.4/ml in supplemented YE medium. The culture was made 1.2 M with respect to sorbitol by adding an equal volume of 2.4 M sorbitol in PBS about 5 minutes prior to fixation. Cells were fixed for 30 minutes at 20°C in 3.8% para-formaldehyde. Cells were washed in PEMS buffer and cell walls were digested by 1.0 mg/ml zymolyase 100T for 70 minutes at 37°C, lysed by 5 minutes incubation in PEMS containing 1% Triton X-100 and then washed three times in PEM buffer before incubation for 30 minutes in PEM buffer with 1% bovine serum albumin, 0.1% sodium azide and 100 mM lysine hydrochloride (PEMBAL) buffer. Rabbit Swi6 antibody was used for overnight incubation with cells in PEMBAL at 4°C. Stained cells were washed three times in PEMBAL and then incubated with secondary antibody for 1h at room temperature. Staining with Hoechst was performed before mounting cells on slides. Microscopy was done on a Nikon Ti confocal microscope using a 100X objective. Image analysis was done in ImageJ. Cells were authenticated by immunostaining.
S. pombe strains used in this study are described in Canzio et al. 2013:
PM0251: P(h+), ura4-DS/E, ade6-M210, leu1–32, imr1L(NcoI)::ura4, otr1R(Sph1)::ade6
DC27: PM0251, swi6Δ:: KanMX:: KanMX, dcr1Δ:: NatMXDC30: PM0251, swi6:: KanMX:: KanMX, dcr1Δ:: NatMX
DC32: PM0251, swi6 (R93A-K94A):: KanMX:: KanMX, dcr1Δ:: NatMX
Sanulli S., Trnka M., Dharmarajan V., Tibble R., Pascal B., Burlingame A., Griffin P., Gross J, & Narlikar G. (2019). HP1 reshapes the nucleosome core to promote phase separation of heterochromatin. Nature, 575(7782), 390-394.
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4

Histological Analysis of Mouse Brains

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A separate cohort of mice was divided into two sub-groups: One sub-group was perfused with PBS, and the other sub-group was perfused with FITC-dextran as previously described31 . PBS-perfused mice brains were embedded with paraffin, and FITC-dextran perfused mice brains were embedded in Optimal Cutting Temperature compound. Tissue blocks were cut into 8 μm (PBS-perfused) and 50 μm (FITC-dextran-perfused) sections. IgG staining was applied to paraffin-embedded sections, and images were recorded using a Leica ICC50 microscope. A Nikon Ti confocal microscope was used for imaging FITC-dextran-perfused frozen sections.
Parchur A.K., Fang Z., Jagtap J.M., Sharma G., Hansen C., Shafiee S., Hu W., Miao Q.R, & Joshi A. (2020). NIR-II Window Tracking of Hyperglycemia Induced Intracerebral Hemorrhage in Cerebral Cavernous Malformation Deficient Mice. Biomaterials science, 8(18), 5133-5144.
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5

Mitochondrial Membrane Potential Assay

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Cultured cells were incubated with 5 nM TMRM (Tetramethylrhodamine methyl ester; Thermo Fisher, cat# T-668) for 30 min at 37 °C. Imaging was carried out using an inverted Nikon Ti confocal microscope using 561 nm excitation. All images were captured using identical microscope settings. Four regions per well were captured and analysis of images was performed by drawing a region of interest around cells at random and measuring mean intensity using ImageJ software. Areas of the image that were absent of cells were used for background correction.
Russell O.M., Fruh I., Rai P.K., Marcellin D., Doll T., Reeve A., Germain M., Bastien J., Rygiel K.A., Cerino R., Sailer A.W., Lako M., Taylor R.W., Mueller M., Lightowlers R.N., Turnbull D.M, & Helliwell S.B. (2018). Preferential amplification of a human mitochondrial DNA deletion in vitro and in vivo. Scientific Reports, 8, 1799.
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6

Immunostaining of S. pombe for Swi6 Localization

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Cells were grown to mid log phase, OD 600nm =0.2–0.4/ml in supplemented YE medium. The culture was made 1.2 M with respect to sorbitol by adding an equal volume of 2.4 M sorbitol in PBS about 5 minutes prior to fixation. Cells were fixed for 30 minutes at 20°C in 3.8% para-formaldehyde. Cells were washed in PEMS buffer and cell walls were digested by 1.0 mg/ml zymolyase 100T for 70 minutes at 37°C, lysed by 5 minutes incubation in PEMS containing 1% Triton X-100 and then washed three times in PEM buffer before incubation for 30 minutes in PEM buffer with 1% bovine serum albumin, 0.1% sodium azide and 100 mM lysine hydrochloride (PEMBAL) buffer. Rabbit Swi6 antibody was used for overnight incubation with cells in PEMBAL at 4°C. Stained cells were washed three times in PEMBAL and then incubated with secondary antibody for 1h at room temperature. Staining with Hoechst was performed before mounting cells on slides. Microscopy was done on a Nikon Ti confocal microscope using a 100X objective. Image analysis was done in ImageJ. Cells were authenticated by immunostaining.
S. pombe strains used in this study are described in Canzio et al. 2013:
PM0251: P(h+), ura4-DS/E, ade6-M210, leu1–32, imr1L(NcoI)::ura4, otr1R(Sph1)::ade6
DC27: PM0251, swi6Δ:: KanMX:: KanMX, dcr1Δ:: NatMXDC30: PM0251, swi6:: KanMX:: KanMX, dcr1Δ:: NatMX
DC32: PM0251, swi6 (R93A-K94A):: KanMX:: KanMX, dcr1Δ:: NatMX
Sanulli S., Trnka M., Dharmarajan V., Tibble R., Pascal B., Burlingame A., Griffin P., Gross J, & Narlikar G. (2019). HP1 reshapes the nucleosome core to promote phase separation of heterochromatin. Nature, 575(7782), 390-394.
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7

Visualizing Mitochondrial Dynamics in Cells

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MEFs or neurons were grown on chamber slides and infected with recombinant adenoviri for 48–72 hours unless otherwise indicated. MEFs were stained with MitoTracker Green (200 nM, Invitrogen M7514) and Hoechst (10mg/ml, Invitrogen H3570), with or without tetramethylrhodamine ethyl ester (TMRE; 200 nM, Invitrogen T-669). Neuronal mitochondria were labeled with adenoviral-expressed mitoGFP plus TMRE, or with adeno-mitoDsRed for time-lapse studies. Cover slips were loaded onto a chamber (Warner instrument, RC-40LP) in modified Krebs-Henseleit buffer (138 mM NaCl, 3.7 mM KCl, 1.2 mM KH2PO4, 15 mM Glucose, 20 mM HEPES and 1 mM CaCl2) at room temperature and visualized on a Nikon Ti Confocal microscope equipped with a 60× 1.3NA oil immersion objective. For mitophagy studies MEFs were stained with MitoTracker Green and infected with adeno-mcherry Parkin or stained with LysoTracker Deep Red (50 nM, Invitrogen L-7528); carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP, 10 µM for 1 hour) was applied as a positive control. Mitochondrial fusion was measured at 2 and 6 hours after PEG-mediated cell fusion of MEFs treated 48 hours previously with adeno-mitoGFP or adeno-mitoDsRed14 (link). Laser confocal fluorescence was excited with 561 nm (MitoTracker Green, GFP, FITC), 637 nm (TMRE, LysoTracker Red, mcherry-Parkin, MitoTracker Orange) or 408 nm (Hoechst) laser diodes.
Franco A., Kitsis R.N., Fleischer J.A., Gavathiotis E., Kornfeld O.S., Gong G., Biris N., Benz A., Qvit N., Donnelly S.K., Chen Y., Mennerick S., Hodgson L., Mochly-Rosen D, & Dorn GW I.I. (2016). Correcting mitochondrial fusion by manipulating mitofusin conformations. Nature, 540(7631), 74-79.
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8

Confocal Microscopy Imaging Protocol

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All images were captured using a Nikon Ti Confocal microscope, using four lasers for fluorescence: 405, 488, 563, and 647 nm. The NISelements (ver 3.22.08, Melville, NY, USA) software was used to set up analyses. The corresponding primary antibody’s negative control was used to control non-specific staining for each batch of analysed slides. In this regard, specific laser intensity and gain parameters were chosen to remove any signal corresponding to 488, 563 and 647 nm. In addition, epifluorescence site conformation was done before confocal imaging to ensure the highest quality of the images.
Odrzywolski A., Jarosz B., Kiełbus M., Telejko I., Ziemianek D., Knaga S, & Rola R. (2021). Profiling Glioblastoma Cases with an Expression of DCX, OLIG2 and NES. International Journal of Molecular Sciences, 22(24), 13217.
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9

Multiparametric Live Cell Imaging of Mitochondria

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Live cell imaging used a Nikon Ti confocal microscope equipped with a 60x 1.3NA oil immersion objective. For visualization of mitochondria and measurement of mitochondrial membrane potential, MEFs were stained with 200 nM MitoTracker Green, 200 nM of tetramethylrhodamine, ethyl ester (TMRE) and 10 μg/ml Hoechst at 37 °C for 30 min. Fluorescence was excited with a 405 nm laser diode (Hoechst), a 488 nm Argon laser (MitoTracker Green) and a 543 nm HeNe laser (TMRE). For detection of parkin aggregation, MEFs were stained with MitoTracker Green and Hoechst; fluorescence for mcherry-parkin was excited with a 543 nm HeNe laser. For assessment of lysosomal engulfed mitochondria, MEFs were stained with 50 nM LysoTracker Red, MitoTracker Green and Hoechst; fluorescence for LysoTracker Red was excited with a 543 nm HeNe laser.
Song M., Mihara K., Chen Y., Scorrano L, & Dorn GW I.I. (2015). Mitochondrial fission and fusion factors reciprocally orchestrate mitophagic culling in mouse hearts and cultured fibroblasts. Cell metabolism, 21(2), 273-285.
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10

Visualization and Quantification of Protein Expression

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The cells were seeded onto 24-well glass-bottom plates (MoBiTec, Goettingen Germany) a day before transfection. Transfection was conducted as described above. Transfected cells were then visualized under a Nikon Ti Confocal microscope after 24 and 28 h since transfection, using a 563 nm laser for mCardinal fluorescence. Mean fluorescence intensity was measured using NISelements (ver 3.22.08, Melville, NY, USA) software and is shown on the graphs. Each of at least 10 measured regions of interest (ROIs) included a cluster of more than 20 adherent cells. The brightest slices of the z-stacks were chosen for measurement. The measurements were done in duplicate.
Kiełbus M., Czapiński J., Kałafut J., Woś J., Stepulak A, & Rivero-Müller A. (2019). Genetically Engineered Lung Cancer Cells for Analyzing Epithelial–Mesenchymal Transition. Cells, 8(12), 1644.
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