Tcs sp8 microscope

Manufactured by Leica Microsystems

Sourced in Germany

The TCS SP8 is a confocal laser scanning microscope designed for high-resolution imaging of biological samples. It features a modular design, allowing for customization to meet specific research needs. The microscope utilizes a multi-channel detection system and a variety of laser options to provide versatile imaging capabilities.

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Lab products found in correlation

Las x software, by Leica Microsystems (3 mentions) Cell culture insert, by Thermo Fisher Scientific (2 mentions) Alexa fluor488 labeled anti rabbit secondary antibody, by Abcam (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions) 8 well plate, by Ibidi (1 mentions) Leica research microscope dm ire2, by Leica Microsystems (1 mentions) Eclipse ti2 microscope, by Nikon (1 mentions) Hoechst 33342, by Cell Signaling Technology (1 mentions) Pkh67, by Merck Group (1 mentions) Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions) Bx51 microscope, by Olympus (1 mentions) Phosphate buffered saline (pbs), by Fujifilm (1 mentions) Tnf receptor 1 polyclonal antibody, by Bioss Antibodies (1 mentions) Rnace it cocktail, by Agilent Technologies (1 mentions) Prolong gold, by Thermo Fisher Scientific (1 mentions) Tcs sp5, by Leica Microsystems (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Dojindo Laboratories (1 mentions) Live dead viability kit, by Thermo Fisher Scientific (1 mentions) Bz x700 fluorescence microscope, by Keyence (1 mentions) Evo 10, by Zeiss (1 mentions)

25 protocols using tcs sp8 microscope

Cellular Internalization of Labeled Peptides

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HeLa cells were seeded into 8-well plates (ibidi, Gräfelfing, Germany, 40,000 cells per well, respectively) and grown to 80–90% confluency. Cells were treated with 1 µM CF-labeled peptide in serum-free medium for 30 min or 2 h at 37 °C. Ten minutes prior to the end of the incubation time, nuclei were stained with Hoechst33342. The peptide solution was removed, and cells were treated with trypan blue (150 mM in 0.1 M acetate buffer, pH 4.15) for 30 s. Cells were washed twice with PBS and covered with fresh medium supplemented with FBS. Microscopic analysis was performed using an inverse confocal TCS SP8 microscope (Leica Microsystems, Wetzlar, Germany), equipped with a 63× oil-immersion objective. Images were recorded with LAS X software (Leica Microsystems, Wetzlar, Germany) and adjusted with Fiji.

Lützenburg T., Burdina N., Scholz M.S, & Neundorf I. (2021). Improving Membrane Activity and Cargo Delivery Efficacy of a Cell-Penetrating Peptide by Loading with Carboranes. Pharmaceutics, 13(12), 2075.

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Fluorescence Microscopy Protocol

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An Olympus microscope BX53 was used to take the fluorescence images containing DAPI staining. A Leica research microscope (DM IRE2) (Leica Microsystems AG, Wetzlar, Germany) was used for taking fluorescence images in Figure 5 and Figure 6. Figure 3E,F were made by confocal microscopy (Leica TCS SP8 microscope and LAS X software, Leica Microsystems, ver. 2.0.2.15022) [81 (link)], using the facilities of the Advanced Neural Imaging Center of the Korean Brain Research Institute (KBRI). We used Adobe Systems Photoshop 7.0 software (Adobe, San Jose, CA, USA) for processing digital images.

Islam M.A., Choi H.J., Dash R., Sharif S.R., Oktaviani D.F., Seog D.H, & Moon I.S. (2020). N-Acetyl-d-Glucosamine Kinase Interacts with NudC and Lis1 in Dynein Motor Complex and Promotes Cell Migration. International Journal of Molecular Sciences, 22(1), 129.

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Fluorescent Labeling of Silk Ionomers

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Carboxylated and aminated silks were fluorescently labeled with FITC to visualize nanolayer deposition on cell surface. Briefly, FITC was dissolved in DMSO at a concentration of 10 mg/mL, diluted 4 times with DI water and 100 μL of it was added dropwise to 10 mL of 5 mg.mL⁻¹ solution of silk ionomers in 0.1 M carbonate buffer (pH 9.0). The mixture was stirred for 1 h at RT and then dialyzed in 3.5 kDa cutoff dialysis membranes against DI water for 3 days with 6 water changes. Solutions were frozen overnight, lyophilized, and stored at −20°C. Fluorescence emission by 0.1 mg.mL⁻¹ of FITC-labeled ionomer solutions at excitation / emission wavelengths of 488 nm / 520 nm was converted to FITC concentration / mg protein using a calibration curve obtained with known concentrations of FITC solutions (Fig.S1B). L929 cells and hMSCs were LbL nanocoated with FITC-labeled SF ionomers and visualized at 2h after seeding and at days 1, 3 and 8 using a TCS SP8 microscope from Leica Microsystems (Wetzlar, Germany) at excitation and emission wavelengths of 488 nm/500–540 nm. Relative fluorescence intensity of silk ionomer layers deposited on individual cells was quantified by mean gray values on Day 0 confocal images using ImageJ.

Hasturk O., Sahoo J.K, & Kaplan D.L. (2020). Synthesis and characterization of silk ionomers for layer-by-layer electrostatic deposition on individual mammalian cells. Biomacromolecules, 21(7), 2829-2843.

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Epithelial-Mesenchymal Transition Dynamics

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Cells were grown and treated with 5 ng·mL⁻¹ TGFβ1 on a glass‐bottom dish, and EMT time‐lapse imaging (Movie S1) was performed with an TCS SP8 microscope (Leica Microsystems, Wetzlar, Germany). The time‐lapse images were acquired every 15 min for a total imaging time of 48 h. After induction of EMT by TGFβ1 for 7 days, the cells were grown without TGFβ1 and MET imaging was performed with an ECLIPSE Ti2 microscope (Nikon, Minato, Japan). Images were acquired every 30 min with a total imaging time of 62 h (Movie S2). Five days after mammosphere formation in ultra‐low attachment condition (3D), mammospheres were grown with (complete EMT) or without (partial EMT) 5 ng·mL⁻¹ TGFβ1 on a glass‐bottom dish, and time‐lapse imaging was performed with an ECLIPSE Ti2 microscope (Nikon, Minato, Japan). Images were acquired every 8 min for a total imaging time of 55 h (Movie S3).
For RFP intensity quantification of migratory cells from mammospheres, 3 cells were selected at random from each quadrant of one sphere in the differential interference contrast (DIC) channel (total 60 cells per condition). Then, the mean RFP intensity was measured in the RFP channel by image j software. All experiments were recorded using identical image acquisition conditions.

Tsubakihara Y., Ohata Y., Okita Y., Younis S., Eriksson J., Sellin M.E., Ren J., ten Dijke P., Miyazono K., Hikita A., Imamura T., Kato M., Heldin C, & Moustakas A. (2022). TGFβ selects for pro‐stemness over pro‐invasive phenotypes during cancer cell epithelial–mesenchymal transition. Molecular Oncology, 16(12), 2330-2354.

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Immunofluorescence Staining of TRPV1 Receptor

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Cells were grown for 24 h on 15 mm glass coverslips in a 12-well plate. Samples were then fixed with 3% PFA in PBS, permeabilized with 0.25% ice-cold TRITON X100 in PBS and blocked with 1% BSA in PBS. The coverslips were stained with a rabbit anti-human TRPV1 antibody (1:250, ab3487-ABCAM; Cambridge, UK) for 1 h at room temperature and then incubated with TRITC-anti rabbit secondary antibody (1:500, ab6718-ABCAM; Cambride, UK) in the dark for 1 h at room temperature. Negative controls were prepared by omitting the primary antibody. Nuclei were stained with 1 µg/mL Hoechst 33342 (#4082-Cell Signaling Technologies; Danvers, MA, USA) and cell membrane with 2 µM PKH67 (MINI67–Sigma-Aldrich; Milan, Italy). Finally, the coverslips were mounted on glass slides using ProLong Gold antifade reagent (Invitrogen, Carlsbad, CA, USA) as mounting medium. Immunofluorescence images were acquired using an Olympus BX51 microscope, whereas the confocal microscopy images were captured using a Leica TCS SP8 Microscope and analyzed with LAS X software (Leica Microsystems GmbH, Wetzlar, Germany).

Faris P., Ferulli F., Vismara M., Tanzi M., Negri S., Rumolo A., Lefkimmiatis K., Maestri M., Shekha M., Pedrazzoli P., Guidetti G.F., Montagna D, & Moccia F. (2020). Hydrogen Sulfide-Evoked Intracellular Ca2+ Signals in Primary Cultures of Metastatic Colorectal Cancer Cells. Cancers, 12(11), 3338.

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Visualizing rhodamine B in silk

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The distribution of rhodamine B molecules in sprayed silk fibers and aerosols was investigated using a TCS SP8 microscope (Leica Microsystems, Wetzlar, Germany).

Hamilton M.L., Van Remmen H., Drake J.A., Yang H., Guo Z.M., Kewitt K., Walter C.A, & Richardson A. (2001). Does oxidative damage to DNA increase with age?. Proceedings of the National Academy of Sciences of the United States of America, 98(18), 10469-10474.

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Visualizing TNFR1 and TNFR2 in ARPE-19 Cells

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ARPE-19 cells were cultured on glass bottom plates (AGC Techno Glass, Shizuoka, Japan) for 24 h, then co-cultured with MT2 and Jurkat cells using cell culture inserts (Thermo Fisher Scientific) for 48 h. After three washes with phosphate-buffered saline (PBS) (Wako Pure Chemical Corporation), ARPE-19 cells were fixed by cold fixing buffer (methanol/acetone, 1:1) at −20°C for 20 min and blocked with 10% FBS in PBS for 15 min. Cells were then incubated in the diluted primary antibodies for 1 h at room temperature, followed by incubation with Alexa fluor488-labeled anti-rabbit secondary antibody (Abcam, Tokyo, Japan) along with 4′,6-diamidino-2-phenylindole dihydrochloride (Cosmo Bio, Tokyo, Japan) incubation for 1 h at room temperature. The following antibodies were used as primary antibodies: TNF receptor 1 polyclonal antibody (Bioss Antibodies, Woburn, MA) and TNF receptor 2 polyclonal antibody (Proteintech, Chicago, IL). We scanned using a TCS-SP8 microscope (Leica Micro Systems, Wetzlar, Germany).

Uchida M., Kamoi K., Ando N., Wei C., Karube H, & Ohno-Matsui K. (2019). Safety of Infliximab for the Eye Under Human T-Cell Leukemia Virus Type 1 Infectious Conditions in vitro. Frontiers in Microbiology, 10, 2148.

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Immunostaining and DNA FISH Analysis

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Paraformaldehyde-fixed cells were stained with macroH2A1.2 antibody as described above. After immunostaining, cells were fixed for the second time with methanol and acetic acid solution (3:1 vol/vol) at room temperature for 10 min, followed by fixation with 2% paraformaldehyde (PFA) for 1 min. Coverslips were treated with RNace-iT cocktail (1:1,000 dilution; Agilent Technologies) at 37°C for 1 h and dehydrated in a 70%, 90%, and 100% ethanol series for 3 min each and air dried. DNA FISH probes were prepared using the FISH-Tag DNA multicolor labeling kit (Life Technologies) according to the manufacturer’s protocol. Hybridization was performed overnight in 1× hybridization buffer (Empire Genomics) with 50 to 75 ng of labeled probe DNA at 37°C. Slides were washed at room temperature with 1× phosphate-buffered detergent (PBD; MP Biosciences), followed by washing with wash buffer (0.5× SSC, 0.1% SDS) at 65°C. Nuclei were stained with DAPI, coverslips were mounted using Prolong Gold (Life Technologies), and images were captured using a TCS-SP5 or TCS-SP8 microscope (Leica Microsystems).

Khurana S., Markowitz T.E., Kabat J, & McBride A.A. (2021). Spatial and Functional Organization of Human Papillomavirus Replication Foci in the Productive Stage of Infection. mBio, 12(6), e02684-21.

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Cellular Internalization of Fluorescent Peptides

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Cells were seeded into 8-well plates (HeLa cells 35,000 and HFF-1 25,000 cells per well, respectively) and grown to 80–90% confluency. Cells were treated with 10 μM CF- or dox-labeled peptides in a serum-free medium for 30 min at 37 °C. Ten minutes prior to the end of the incubation time, nuclei were stained with Hoechst33342. The peptide solution was removed, and cells were washed five times with PBS and covered with fresh medium including FBS. Microscopic analysis was performed using an inverse confocal TCS SP8 microscope (Leica Microsystems, Wetzlar, Germany), equipped with a 63× oil-immersion objective. Images were recorded with LAS X software (LAS_X_Core_3.7.6_25997, Leica Microsystems, Wetzlar, Germany). For the GUV analysis and the uptake of the peptide-drug conjugates the Keyence fluorescence tabletop microscope BZ-X800E (Keyence, Osaka, Japan), with a 60× oil immersion lens was used. Images were recorded with Keyence software (BZ-800X_Analyzer 1.1.1.8, Keyence, Osaka: Japan) and evaluated with Fiji.

Grabeck J., Lützenburg T., Frommelt P, & Neundorf I. (2022). Comparing Variants of the Cell-Penetrating Peptide sC18 to Design Peptide-Drug Conjugates. Molecules, 27(19), 6656.

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Immunofluorescence Staining of Cultured Cells

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Cells fixed with 3.7% paraformaldehyde in PBS were permeabilized by incubating with 0.5% Triton X-100 in PBS for 5 min. After blocking with 3% skim milk in PBS for 30 min, the cells were incubated with antibodies at 4 °C overnight, washed five times with PBS, and then incubated with Alexa Fluor 488- or 594-conjugated anti-mouse or anti-rabbit antibodies (1: 100 dilution) at 25 °C for 1 h. Nuclei were counterstained with 100 ng/mL DAPI (Dojindo Laboratories) for 10 min. The cells were observed under a Leica Microsystems TCS SP8 microscope.

Higa M., Oka M., Fujihara Y., Masuda K., Yoneda Y, & Kishimoto T. (2018). Regulation of inflammatory responses by dynamic subcellular localization of RNA-binding protein Arid5a. Proceedings of the National Academy of Sciences of the United States of America, 115(6), E1214-E1220.

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