Tcs sp8 sted 3x confocal microscope

Manufactured by Leica

Sourced in Germany

The Leica TCS SP8 STED 3X is a confocal microscope system designed for advanced fluorescence imaging. It utilizes Stimulated Emission Depletion (STED) technology to achieve super-resolution capabilities beyond the diffraction limit of light. The system provides high-speed, high-resolution imaging with a versatile range of laser excitation wavelengths and flexible detector configurations.

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Lab products found in correlation

Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (4 mentions) Paraformaldehyde (pfa), by Electron Microscopy Sciences (3 mentions) Sodium arsenite, by Merck Group (2 mentions) 8 well glass slides, by Merck Group (2 mentions) Fugene 6, by Promega (2 mentions) Alexa fluor 488 conjugate, by Thermo Fisher Scientific (2 mentions) Mouse anti cd63 e 12, by Santa Cruz Biotechnology (2 mentions) Rabbit anti shh h 160, by Santa Cruz Biotechnology (2 mentions) Rat anti ha, by Roche (2 mentions) Prism 7, by GraphPad (2 mentions) Tetraspeck fluorescent beads, by Thermo Fisher Scientific (2 mentions) Lightning module, by Leica (2 mentions) Anti pml, by Santa Cruz Biotechnology (1 mentions) Na 1.4 oil immersion objective, by Leica (1 mentions) Eif4g sc 11373, by Santa Cruz Biotechnology (1 mentions) Alexa fluor 488, by Thermo Fisher Scientific (1 mentions) Ab183136, by Abcam (1 mentions) Alexa fluor 488 or 555, by Thermo Fisher Scientific (1 mentions) Parafolmaldehyde, by Electron Microscopy Sciences (1 mentions) Sc 16377, by Santa Cruz Biotechnology (1 mentions)

30 protocols using tcs sp8 sted 3x confocal microscope

Visualizing EV Uptake in Recipient Cells

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To examine the uptake of EVs by recipient cells, the EVs purified from HEK293T cells were stained with the red fluorescent dye PKH26 (Sigma) for 5 min at room temperature, and neutralized using exosome-free FBS. The PKH26-labelled EVs were washed with PBS and centrifuged at 100,000 g for 2 h at 4°C. Huh7 cells were incubated with the resuspended PKH26-labelled EVs for 3 h, followed by washing with PBS. 4% paraformaldehyde (PFA) was used to fix cells at room temperature for 15 min, and Hoechst 33342 (Beyotime) was used to stain the nucleus of cells. The uptake of EVs by Huh7 cells was observed using the TCS-SP8 STED 3X confocal microscope (Leica).
For immunocytofluorescence analysis, cells were fixed in 4% PFA for 15 min and permeabilized with 0.5% TritonX-100 for 15 min, followed by incubation with anti-CD63 antibody (Abcam) overnight at 4°C. Cells were stained with Hoechst 33342 (Beyotime) and observed using the TCS-SP8 STED 3X confocal microscope (Leica).

Zeng W., Zheng L., Li Y., Yang J., Mao T., Zhang J., Liu Y., Ning J., Zhang T., Huang H., Chen X, & Lu F. (2023). Engineered extracellular vesicles for delivering functional Cas9/gRNA to eliminate hepatitis B virus cccDNA and integration. Emerging Microbes & Infections, 13(1), 2284286.

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Immunofluorescence and Q-FISH on FFPE Tissue

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FFPE tissue samples were fixed in 10% buffered formalin, dehydrated, embedded in paraffin wax and sectioned at 4 μm. Tissue sections were deparaffinized in xylene and re-hydrated through a series of decreasing ethanol concentrations up to water. Immunofluorescence (IF) was performed on deparaffined tissue sections processed with 10 mM sodium citrate (pH 6.5) cooked under pressure for 2 min. Tissue sections were permeabilized with 0.5% Triton in PBS and blocked with 5% BSA in PBS. Samples were incubated overnight at 4 ºC with rabbit polyclonal anti-PML (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA, H-238). Q-FISH was performed on IF-stained slides fixed with 4% formaldehyde for 20 min. The DAPI images were used to detect telomeric signals inside each nucleus. Immunofluorescence images were obtained with a TCS-SP8 STED 3X confocal microscope equipped with a 63×/NA 1.4 oil immersion objective, a white light laser and LAS X v3.5 software (Leica-Microsystems). Z-stacks of the samples were acquired and then analyzed with Definiens Developer XD 64 v2.5 software (Definiens Inc., Munich, Bayern, Germany).

Monteagudo M., Martínez P., Leandro-García L.J., Martínez-Montes Á.M., Calsina B., Pulgarín-Alfaro M., Díaz-Talavera A., Mellid S., Letón R., Gil E., Pérez-Martínez M., Megías D., Torres-Ruiz R., Rodriguez-Perales S., González P., Caleiras E., Jiménez-Villa S., Roncador G., Álvarez-Escolá C., Regojo R.M., Calatayud M., Guadalix S., Currás-Freixes M., Rapizzi E., Canu L., Nölting S., Remde H., Fassnacht M., Bechmann N., Eisenhofer G., Mannelli M., Beuschlein F., Quinkler M., Rodríguez-Antona C., Cascón A., Blasco M.A., Montero-Conde C, & Robledo M. (2021). Analysis of Telomere Maintenance Related Genes Reveals NOP10 as a New Metastatic-Risk Marker in Pheochromocytoma/Paraganglioma. Cancers, 13(19), 4758.

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Microscopy Protocol for Cytoskeletal Imaging

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After immunofluorescent staining for Ki67, Cleaved Caspase-3, or the tight junction protein, ZO-1, images were acquired on a TCS SP8 STED3x confocal microscope (Leica Microsystems) utilizing a 40x/1.3 oil immersion objective for Cleaved Caspase-3 (zoom 1.25) and a 63x/1.40 oil immersion objective for Ki67 (zoom 1.25) and ZO1 (zoom 3.25). ZO-1 images were processed via morphological segmentation plug-in (ImageJ) for tortuosity calculation, also known as the zigzag index^{23 (link),37 (link),42 (link),43 (link)}. The measured tortuosity was the ratio of segment length and Euclidian distance between two defined ZO-1 segment points^{23 (link)}.

Crawford C.K., Beltran A., Castillo D., Matloob M.S., Uehara M.E., Quilici M.L., Cervantes V.L, & Kol A. (2023). Fenofibrate reduces glucose-induced barrier dysfunction in feline enteroids. Scientific Reports, 13, 22558.

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Subcellular Localization of UBQLN2 Variants

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HeLa cells were seeded on 8-well glass slides (Millipore). Cells were transfected 24 hours after seeding using FuGene 6 (Promega) with mCherry-UBQLN2-Full-Length (wild-type), mCherry- UBQLN2-ΔUBA, mCherry-UBQLN2–450-624, or mCherry-UBQLN2–487-624 construct. 24 hr post transfection, cells were stressed with 500 µM sodium arsenite (Sigma-Aldrich) for 30 min. Cells were then fixed with 4% paraformaldehyde (Electron Microscopy Sciences), permeabilized with 0.5% Triton X-100, and blocked in 5% bovine serum albumin (BSA). Primary antibody used was against eIF4G (sc-11373; Santa Cruz). For visualization, the appropriate host-specific Alexa Fluor 488 (Molecular Probes) secondary antibody was used. Slides were mounted using Prolong Gold Antifade Reagent with DAPI (Life Technologies). Images were captured using a Leica TCS SP8 STED 3X confocal microscope (Leica Biosystems) with a 63x objective. Three independent experiments were performed.

Dao T.P., Kolaitis R.M., Joo Kim H., O’Donovan K., Martyniak B., Colicino E., Hehnly H., Taylor J.P, & Castañeda C.A. (2018). Ubiquitin modulates liquid-liquid phase separation of UBQLN2 via disruption of multivalent interactions. Molecular cell, 69(6), 965-978.e6.

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Actinomycin D-Induced Stress Response

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Transfected HeLa^DL-KO cells were stressed or not with 5 μg/ml actinomycin D for 3 h. Cells were then fixed with 4% parafolmaldehyde (Electron Microscopy Sciences, #15713-S), permeabilized with 0,2% Triton X-100 and blocked with 1% bovine serum albumin (BSA). Primary antibodies used were against G3BP (611127; BD Biosciences), nucleolin (sc-8031; Santa Cruz) and hnRNPDL (ab183136; Abcam). For visualization, the appropriate host-specific Alexa Fluor 488 or 555 (Molecular Probes) secondary antibodies were used. Slides were mounted using ProLong Gold antifade reagent with DAPI (Invitrogen). Images were captured using a Leica TCS SP8 STED 3x confocal microscope (Leica Biosystems) with a 63x oil objective.

Batlle C., Yang P., Coughlin M., Messing J., Pesarrodona M., Szulc E., Salvatella X., Kim H.J., Taylor J.P, & Ventura S. (2020). hnRNPDL Phase Separation Is Regulated by Alternative Splicing and Disease-Causing Mutations Accelerate Its Aggregation. Cell Reports, 30(4), 1117-1128.e5.

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Imaging Fluorescent Peptide Localization

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Prior to peptide treatment, cells were seeded onto 8-well chamber slides. After 24h incubation, DMSO solubilized Alexa Fluor 647 labeled peptides were added to medium for 3 or 4 hours. Then, cells were fixed with 4% paraformaldehyde in PBS for 10 min at room temperature, permeabilized with 0.2% Triton X-100 in PBS for 10 min at room temperature, and then blocked with 10% normal goat serum for 1 h at room temperature. Samples were incubated with primary antibodies in blocking buffer overnight at 4°C. Sam ples were then washed three times with PBS and incubated with secondary antibody for 1 h at room temperature. Secondary antibodies were conjugated with Alexa Fluor 488/555 (Life Technologies). For microscopic imaging, slides were mounted with ProLong Gold Antifade Mountant with DAPI. Images were captured using a Leica TCS SP8 STED 3x confocal microscope (Leica Biosystems) with a 63x oil objective.

White M.R., Mitrea D.M., Zhang P., Stanley C.B., Cassidy D.E., Nourse A., Phillips A.H., Tolbert M., Taylor J.P, & Kriwacki R.W. (2019). C9orf72 poly(PR) Dipeptide Repeats Disturb Biomolecular Phase Separation and Disrupt Nucleolar Function. Molecular cell, 74(4), 713-728.e6.

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Visualizing Stress Granule Dynamics

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HeLa cells were seeded on 8-well glass slides (Millipore). Cells were transfected 24 h after seeding using FuGENE 6 (Promega) with either EGFP-tagged or EYFP-tagged hnRNPA1 constructs. 24 h post transfection, cells were stressed with 500 μM sodium arsenite (Sigma-Aldrich) for 30 min. Cells were then fixed with 4% paraformaldehyde (Electron Microscopy Sciences), permeabilized with 0.5% Triton X-100, and blocked in 5% bovine serum albumin (BSA). Primary antibodies used were against G3BP1 (611127; BD Biosciences) and eIF3η (sc-16377; Santa Cruz). For visualization, the appropriate host-specific Alexa Fluor 555 or 647 (Molecular Probes) secondary was used. Slides were mounted using Prolong Gold Antifade Reagent with DAPI (Life Technologies). Images were captured using a Leica TCS SP8 STED 3X confocal microscope (Leica Biosystems) with a 63X objective. Fluorescent images were quantified by automated puncta analysis using CellProfiler software (Broad Institute). Cells were segmented using DAPI and G3BP1 channels and granules were identified using both G3BP1 and eIF3η channels. Integrated intensities of nucleus, cytoplasm, and granules were measured.
Yeast cells that had been transformed with the indicated GFP-tagged hnRNPA1 protein or vector were grown for 8 h in galactose-containing media, pelleted, and imaged using a Leica-DMIRBE microscope before being processed using ImageJ (NIH).

Beijer D., Kim H.J., Guo L., O’Donovan K.J., Mademan I., Deconinck T., Schil K.V., Fare C., Drake L.E., Ford A., Kochański A., Kabzińska D., Dubuisson N., Bergh P.V.d., Voermans N.C., Lemmers R., Maarel S.v.d., Bonner D., Sampson J., MT W., Mehrabyan A., Palmer S., Jonghe P.D., Shorter J., Taylor J., & Baets J. (2021). DEFINING THE DIVERSITY OF HNRNPA1 MUTATIONS IN CLINICAL PHENOTYPE AND PATHOMECHANISM.

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Quantifying Cardiac Oxidative Stress

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Cardiac level of ROS was determined by using two independent methods. As we previously described 21 (link), O₂^- level in myocardium was detected with dihydroethidium (DHE) fluorescent stain of LV frozen sections (5 μm) by incubation of DHE (5 µM for 1 h at 37 °C). Images were digitized (×400 magnification, 6 fields per heart) under a fluorescence microscope (DP72; Olympus, Tokyo, Japan) with excitation/emission at 488/610 nm. In addition, chloromethyl derivative CM-H2DCFDA (DCF) fluorescent staining of isolated adult mouse cardiomyocytes was used to test ROS level in the myocardium. Cardiomyocytes isolated through Langendorff perfusion system were incubated with 5 µM DCF for 30 min at 37 ℃. Images were obtained using a Leica TCS SP8 STED 3X confocal microscope with 40× 1.3 NA oil immersion objective (excitation 488 nm, emission 525 nm) with fixed scanning parameters. An average of 50 cells of each heart were analyzed for the intensity of DCF fluorescence in ImageJ software.

She G., Du J.C., Wu W., Pu T.T., Zhang Y., Bai R.Y., Zhang Y., Pang Z.D., Wang H.F., Ren Y.J., Sadoshima J., Deng X.L, & Du X.J. (2023). Hippo pathway activation mediates chemotherapy-induced anti-cancer effect and cardiomyopathy through causing mitochondrial damage and dysfunction. Theranostics, 13(2), 560-577.

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Resolution Measurement Approaches for Microscopy

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Resolution measurements in wide-field, deconvolved wide-field, and SIM images acquired by the Elyra PS.1 microscope was calculated according to Sparrow [41 (link)], where the resolution is defined as the minimal distinguishable distance between the intensity maximums of two structures [49 (link)] (Figure 5). To use this approach for PALM the Gauss display must be used, as distances between centroids are not related to localization precision and hence resolution. Similarly, the resolution measurements in the confocal, STED, and STED + deconvolution images obtained by the Leica TCS SP8 STED 3X confocal microscope were also done according to Sparrow [41 (link)].

Kubalová I., Němečková A., Weisshart K., Hřibová E, & Schubert V. (2021). Comparing Super-Resolution Microscopy Techniques to Analyze Chromosomes. International Journal of Molecular Sciences, 22(4), 1903.

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Immunofluorescence of CD3 and CCDC134

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Primary CD8⁺ T cells on cover glasses were washed with ice-cold PBS and fixed with acetone for 5 min. After blocking with PBS with 2% FBS for 30 min, the cells were incubated with rat anti-CD3ϵ antibody (CD3-12) and rabbit anti-CCDC134 antibody (ab106442) overnight at 4°C, and then incubated with TRITC-labeled anti-rat and FITC-conjugated anti-rabbit secondary antibodies for 30 min at 4°C. Samples were then washed and mounted in antifade reagent containing 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) for nuclei staining. After washing, images were observed and acquired using Leica TCS-SP8 STED 3X confocal microscope.

Zhang T., Shi Q., Gu H., Yu B., Yin S., Ge Q., Mo X., Liu X, & Huang J. (2023). CCDC134 facilitates T cell activation through the regulation of early T cell receptor signaling. Frontiers in Immunology, 14, 1133111.

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