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Tcs sp8 confocal system

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The Leica TCS SP8 is a high-performance confocal system designed for advanced microscopy applications. It features a fast and efficient scanner, a selection of lasers, and sensitive detectors to provide high-quality, high-resolution imaging. The system's core function is to enable researchers to capture detailed, three-dimensional images of samples through optical sectioning and confocal microscopy techniques.

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Lab products found in correlation

Application suite x software, by Leica (4 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions) Volocity software, by PerkinElmer (4 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions) Rna ish tissue assay kits, by Thermo Fisher Scientific (3 mentions) Las af software, by Leica (2 mentions) Las x life science software, by Leica (2 mentions) Fluorescence conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions) Hoechst 33342, by Thermo Fisher Scientific (2 mentions) Superfrost plus, by BD (2 mentions) Prolong gold antifade mounting reagent, by Thermo Fisher Scientific (2 mentions) Triton x 100, by Avantor (2 mentions) Alexa fluor 488 or 594 phalloidin, by Thermo Fisher Scientific (1 mentions) Dithiothreitol (dtt), by Merck Group (1 mentions) Las x software, by Leica (1 mentions) Alexa fluor 633 phalloidin, by Thermo Fisher Scientific (1 mentions) Application suite las software, by Leica (1 mentions) Orange g dye, by Merck Group (1 mentions) Dibutyryl cyclic amp, by Merck Group (1 mentions) Igf 1, by Thermo Fisher Scientific (1 mentions)

81 protocols using tcs sp8 confocal system

1

Quantitative Analysis of Immune Cell Dynamics

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Images were acquired with a Leica TCS SP8 confocal system (Leica Microsystems) using the LSA AF software. Images were acquired with a 10X objective with 0.25NA or 20X objective with 0.70NA, with a resolution of 512×512 or 1024×1024 resolution with a z-step of 4µm. Quantitative assessments were performed using FIJI software (NIH) or Imaris software (Bitplane). Density of injected T cells or dendritic cells was determined by dividing the number of labeled T cells/DCs per section by the area of the lymph node section. Beads coverage was quantified by dividing the area occupied by the beads over the area of the lymph nodes. Every other section covering the totality of the lymph nodes were quantified (Around 15 to 20 section per lymph nodes). Lymphatic ablation was measure either by dividing the area occupied by Lyve-1 immunostaining by the area of the sinus or by dividing the total length of lymphatics by the total length of the sinuses. Macrophages size was determined by averaging the Iba1+ area of 10 individual cells randomly chosen per animal.
Statistical analyses were performed using GraphPad Prism software. Specific statistical tests are presented in the text for each experiment. Outlier samples were eliminated using Grubbs’ test with a significance level of 0.05. No estimate of variation between groups was performed.
Louveau A., Herz J., Alme M.N., Salvador A.F., Dong M.Q., Viar K.E., Herod G., Knopp J., Setliff J., Lupi A.L., Mesquita S.D., Frost E.L., Gaultier A., Harris T.H., Cao R., Hu S., Lukens J.R., Smirnov I., Overall C.C., Oliver G, & Kipnis J. (2018). CNS lymphatic drainage and neuroinflammation are regulated by meningeal lymphatic vasculature. Nature neuroscience, 21(10), 1380-1391.
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2

Imaging and Quantification of Lymphatic Immune Cells

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Images were acquired with a Leica TCS SP8 confocal system (Leica Microsystems) using the LAS AF Software. For the images of the complete whole mount, images were acquired with a 10× objective with 0.25 NA. Other confocal images were acquired using a 20× objective with 0.70 NA or a 40× oil immersion objective with 1.30 NA. All images were acquired with at a 512×512 pixel resolution and with a z-step of 4μm. Quantitative assessments were performed using FIJI software (NIH). Percentage of luminal T cells was determined by counting the number of T cells with luminal localization in the sinuses area. T cell density was established by dividing the number of T lymphocytes by the area of meninges. Prox1-positive cell density was defined by dividing the number of Prox1 nuclei by the area of lymphatic vessels. Statistical analyses were performed using GraphPad Prism software. Specific statistical tests are presented in the text for each experiment. Outlier samples were eliminated using the Grubbs’ test with a significance level of 0.01 (only for the rh-VEGF-c experiment). No estimate of variation between groups was performed.
Louveau A., Smirnov I., Keyes T.J., Eccles J.D., Rouhani S.J., Peske J.D., Derecki N.C., Castle D., Mandell J.W., Kevin S.L., Harris T.H, & Kipnis J. (2015). Structural and functional features of central nervous system lymphatics. Nature, 523(7560), 337-341.
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3

Adherens Junction Dynamics Regulation

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Cells were seeded on coverslips and grown to confluence, then cells were either treated with standard medium, starved for 8 h, or treated with 5 μM Rapamacyn for 24 h or 10 mM 4-dithiothreitol (DTT; Sigma-Aldrich) for 3 h. Immunofluorescence staining was then carried out as above described. Alexa Fluor 488 or 594 Phalloidin (Thermo Fisher Scientific) was used to probe F-actin. Images were captured with TCS-SP8 Confocal System (Leica Microsystems) and analyzed with Fiji/ImageJ software (Schneider et al., 2012 (link)), similarly, to what previously described (Di Russo et al., 2017 (link)). Briefly, adherens junctions, considered as the E-cadherin/beta-catenin positive cell-cell border fragment, were measured on the focal plane corresponding to the maximum intensity of the E-cadherin signal. Adherens junctions ratio was calculated as the length of adherens-junctions over the length of the perimeter of the cell that was manually measured by using the border of phalloidin maximum intensity projections. Experiments were performed in triplicate and, overall, at least 100 cells per sample were measured.
Time-lapse images of cells were captured every 20 min for 20 h by AF6000 microscope system and LASX software (Leica Microsystems).
Damiano V., Spessotto P., Vanin G., Perin T., Maestro R, & Santarosa M. (2020). The Autophagy Machinery Contributes to E-cadherin Turnover in Breast Cancer. Frontiers in Cell and Developmental Biology, 8, 545.
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4

Imaging and Quantification of Lymphatic Immune Cells

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Images were acquired with a Leica TCS SP8 confocal system (Leica Microsystems) using the LAS AF Software. For the images of the complete whole mount, images were acquired with a 10× objective with 0.25 NA. Other confocal images were acquired using a 20× objective with 0.70 NA or a 40× oil immersion objective with 1.30 NA. All images were acquired with at a 512×512 pixel resolution and with a z-step of 4μm. Quantitative assessments were performed using FIJI software (NIH). Percentage of luminal T cells was determined by counting the number of T cells with luminal localization in the sinuses area. T cell density was established by dividing the number of T lymphocytes by the area of meninges. Prox1-positive cell density was defined by dividing the number of Prox1 nuclei by the area of lymphatic vessels. Statistical analyses were performed using GraphPad Prism software. Specific statistical tests are presented in the text for each experiment. Outlier samples were eliminated using the Grubbs’ test with a significance level of 0.01 (only for the rh-VEGF-c experiment). No estimate of variation between groups was performed.
Louveau A., Smirnov I., Keyes T.J., Eccles J.D., Rouhani S.J., Peske J.D., Derecki N.C., Castle D., Mandell J.W., Kevin S.L., Harris T.H, & Kipnis J. (2015). Structural and functional features of central nervous system lymphatics. Nature, 523(7560), 337-341.
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5

Confocal Imaging of Transfected Cardiomyocytes

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The TCS SP8 confocal system (Leica Microsystems, Wetzlar, Germany) was used in combination with Application Suite X software (Leica Microsystems) for the confocal fluorescence microscopy of the transfected cells. DAPI was excited at 405 nm and the emission was detected in the range between 410 and 460 nm. Enhanced yellow fluorescent protein (EYFP) was excited at 488 nm and the emission was detected in the range between 493 and 560 nm. Texas Red was excited at 552 nm and the emission was detected in the range between 570 and 781 nm. Cy3 was excited at 552 nm and the emission was detected in the range between 556 and 758 nm. Excitation and emission detection for the three fluorescence channels was sequentially performed. Then, 3D stacks were imaged for hiPSC-derived cardiomyocytes and presented as intensity projections.
Brodehl A., Holler S., Gummert J, & Milting H. (2022). The N-Terminal Part of the 1A Domain of Desmin Is a Hot Spot Region for Putative Pathogenic DES Mutations Affecting Filament Assembly. Cells, 11(23), 3906.
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6

Intracellular Actin Staining in Lipid Rafts

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In a subset of samples, at the end of the lipid raft labelling procedure, the intracellular actin staining procedure was performed. Briefly, cells were fixed by cold 4% paraformaldehyde in PBS for 20 min. After membrane permeabilization by 0.1% Triton X-100, coverglasses were incubated with PBS containing 2% bovine serum albumin as unspecific site blocking agent and, in turn, with Alexa Fluor 633 phalloidin (Thermo Fisher Scientific) (dilution 1:400) as total actin labelling agent. After 3 washes by 0.1% Tween in PBS coverslips were mounted using glycerol and stored at +4 °C until analysis by confocal microscopy. A three-dimensional scan of labeled cells was performed by the TCS-SP8 Confocal System (Leica Microsystems) interfaced with the Leica Application Suite software. Confocal cell imaging settings were as above. By ImageJ software (National Institutes of Health), mean fluorescence intensity was evaluated in each image of the collected z-stack in manually defined regions of interest.
Agostini F., Vicinanza C., Biolo G., Spessotto P., Da Ros F., Lombardi E., Durante C, & Mazzucato M. (2021). Nucleofection of Adipose Mesenchymal Stem/Stromal Cells: Improved Transfection Efficiency for GMP Grade Applications. Cells, 10(12), 3412.
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7

Quantitative Analysis of Immune Cell Dynamics

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Images were acquired with a Leica TCS SP8 confocal system (Leica Microsystems) using the LSA AF software. Images were acquired with a 10X objective with 0.25NA or 20X objective with 0.70NA, with a resolution of 512×512 or 1024×1024 resolution with a z-step of 4µm. Quantitative assessments were performed using FIJI software (NIH) or Imaris software (Bitplane). Density of injected T cells or dendritic cells was determined by dividing the number of labeled T cells/DCs per section by the area of the lymph node section. Beads coverage was quantified by dividing the area occupied by the beads over the area of the lymph nodes. Every other section covering the totality of the lymph nodes were quantified (Around 15 to 20 section per lymph nodes). Lymphatic ablation was measure either by dividing the area occupied by Lyve-1 immunostaining by the area of the sinus or by dividing the total length of lymphatics by the total length of the sinuses. Macrophages size was determined by averaging the Iba1+ area of 10 individual cells randomly chosen per animal.
Statistical analyses were performed using GraphPad Prism software. Specific statistical tests are presented in the text for each experiment. Outlier samples were eliminated using Grubbs’ test with a significance level of 0.05. No estimate of variation between groups was performed.
Louveau A., Herz J., Alme M.N., Salvador A.F., Dong M.Q., Viar K.E., Herod G., Knopp J., Setliff J., Lupi A.L., Mesquita S.D., Frost E.L., Gaultier A., Harris T.H., Cao R., Hu S., Lukens J.R., Smirnov I., Overall C.C., Oliver G, & Kipnis J. (2018). CNS lymphatic drainage and neuroinflammation are regulated by meningeal lymphatic vasculature. Nature neuroscience, 21(10), 1380-1391.
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8

Immunofluorescence Microscopy Protocol for LC3B Labeling

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For immunofluorescence analyses, cells were cultured on coverslips in the medium described in the text. After treatment, cells were fixed in 4% paraformaldehyde/PBS for 15 min, permeabilized with 0.4% Triton X-100/PBS for 10 min, and incubated for 1 h with 0.04% Triton X-100/PBS containing 3% of BSA to block non-specific antigens. In the case of antibody-mediated LC3B labeling, cells were permeabilized with ice-cold 100% methanol. Cells were stained with the appropriate antibody (reported in Supplementary Table 2) at 4°C overnight. Primary antibodies were visualized with goat anti-mouse Alexa Fluor 594 or 633 (Thermo Fisher Scientific), or with goat anti-rabbit Alexa Fluor 594 or 633 (Thermo Fisher Scientific). TO-PRO-3 (Thermo Fisher Scientific) was used for nuclear labeling. Cells were then analyzed by using the TCS-SP8 Confocal System (Leica Microsystems) interfaced with the Leica Application Suite (LAS) software. When all three fluorochromes were used, individual cells were identified by capturing transmitted light images. Total and colocalized dots were quantified by using the Fiji/ImageJ software (Schneider et al., 2012 (link)) and the ComDet v.0.5.1 plugin1.
Damiano V., Spessotto P., Vanin G., Perin T., Maestro R, & Santarosa M. (2020). The Autophagy Machinery Contributes to E-cadherin Turnover in Breast Cancer. Frontiers in Cell and Developmental Biology, 8, 545.
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9

Fluorescence Microscopy of Transfected Cells

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HeLa and HEK293T adhered to sterile cover slips were transfected with pcDNA3.1 based ERG constructs as described above. Forty-eight hours after transfection cells were fixed and stained by primary and secondary antibodies (listed in Table C in S1 File) and by DAPI (Thermo Fisher Scientific). Microscope slides were inspected using Leica DMi8 inverted microscope equipped with TCS SP8 confocal system and Leica Application Suite X software (Leica Microsystems, Germany).
Potuckova E., Zuna J., Hovorkova L., Starkova J., Stary J., Trka J, & Zaliova M. (2016). Intragenic ERG Deletions Do Not Explain the Biology of ERG-Related Acute Lymphoblastic Leukemia. PLoS ONE, 11(8), e0160385.
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10

Dual-channel Confocal Imaging of Chlorophyll

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Images were acquired with a Leica TCS SP8 confocal system (Leica Microsystems) and the LAS X Life Science Software, while using a HC PL APO ×40/1.10 objective. All images were acquired at a 4096×4096-pixel resolution, with emission at 500-520 nm following excitation at 488nm for chl-roGFP2
fluorescence and emission at 670nm following excitation at 488nm for chlorophyll fluorescence. Merged images were generated using Fiji (Image 1.A) software.
Hipsch M., Lampl N., Zelinger E., Barda O., & Rosenwasser S. (2020). Sensing stress responses in potato with whole-plant redox imaging.
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