Lsm 510 uv confocal microscope

Manufactured by Zeiss

Sourced in Germany

The Zeiss LSM 510 UV confocal microscope is a high-performance imaging system designed for advanced fluorescence microscopy. It utilizes a UV laser source to enable imaging of a wide range of fluorophores, providing researchers with the ability to visualize and analyze samples with exceptional clarity and resolution.

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20 protocols using lsm 510 uv confocal microscope

Measurement of Macromolecular Synthesis in Embryos

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Embryos were cultured in KSOM containing 10 μM 5-ethynyl-2′-deoxyuridine (EdU) or 1 mM ethynyl uridine (EU) for 1 h or 20 μM O-propargylpuromycin (OPP) for 3 h and fixed in 4% paraformaldehyde in PBS for 25 min at room temperature. Time of fixation in all cases was at 48 h after hCG administration. EdU, EU or OPP incorporation into DNA, RNA or newly translated proteins, respectively, was detected using Click-iT® EdU Alexa Fluor® 488 Imaging Kit, Click-iT® RNA Alexa Fluor® 488 Imaging Kit, or Click-iT® Plus OPP Alexa Fluor® 488 Protein Synthesis Assay Kit (Thermo-Fisher), according to the manufacturer’s protocols. Fluorescence was detected on a Zeiss LSM 510 UV confocal microscope.

Gambini A., Stein P., Savy V., Grow E.J., Papas B.N., Zhang Y., Kenan A.C., Padilla-Banks E., Cairns B.R, & Williams C.J. (2020). Developmentally programmed tankyrase activity upregulates β-catenin and licenses progression of embryonic genome activation. Developmental cell, 53(5), 545-560.e7.

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Immunofluorescence Staining of Adherent Cells

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Indicated cells were plated onto sterile Fisherbrand microscope cover glass slips (ThermoFisher Scientific) and placed in Corning Costar Flat Bottom six‐well Cell Culture Plates (Corning Inc). The cells were allowed to grow to 80% confluence, washed three times with phosphate buffered saline (PBS), and fixed with 4% paraformaldehyde for 20 minutes. After fixation, the coverslips were transferred to a clean six‐well plate, and cells were again washed with PBS. Cells were then incubated with 0.1% saponin‐TBST for 10 minutes at 37°C, after which they were washed three times in 0.1% saponin‐TBST. Cells were then blocked for 30 minutes using a background sniper (BIOCARE Medical, Pacheco, CA). Following the blocking step, the cells were washed and incubated with SBP1 primary antibody (MBL) overnight at 1:150 diluted in Diamond Antibody Diluent (Cell Marque, Rocklin CA) in a humid chamber to prevent drying. Cells were then washed three times in 0.1% saponin‐TBST. Secondary antibody (Alexafluor‐647) was then incubated at 1:200 in Diamond Antibody Diluent for 1 hour at room temperature in a dark, humid chamber. Cells were then washed three times in 0.1% saponin‐TBST, after which they were washed three times in PBS. Cells were mounted using ProLong Gold Antifade reagent with 4′,6‐diamidino‐2‐phenylindole (Invitrogen). Images were obtained using an LSM510UV confocal microscope (Zeiss).

Elhodaky M., Hong L.K., Kadkol S, & Diamond A.M. (2020). Selenium‐binding protein 1 alters energy metabolism in prostate cancer cells. The Prostate, 80(12), 962-976.

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Subcellular Localization of TRAF Proteins

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Subconfluent Neuro2a cells grow on cover slips. Cells were then rinsed with PBS buffer at pH 7.4 and fixed in ice-cold methanol for 10 min at −20 °C. After 45-min incubation in 10 % FBS to block nonspecific protein binding, the fixed cells were incubated with the following primary antibodies anti-TRAF2 (sc-876) or anti-TRAF3 (sc-949) diluted (1:300) in PBS containing 3 % low-fat milk at 37 °C for 2 h. After washing with PBS containing 3 % low-fat milk, the fixed cells were incubated with FITC-conjugated anti-IgG antibody (1:150, Sigma-Aldrich) diluted in PBS containing 3 % low-fat milk, at 37 °C for 1 h. After washing, the cover slips were mounted on slides using Prolong Gold antifade reagent (Invitrogen). Fluorescence microscopy was performed using a Zeiss LSM510/UV confocal microscope with a ×63 oil immersion objective. Immuno-labeled slides (n = 4–5 representative fields per slide) were sectioned optically at 0.5-μm intervals (one focal plane) through the cell monolayer to obtain the appropriate focal depth. Images were captured and collected using Axiovision 4.7 program.

El Hokayem J., Brittain GC I.V., Nawaz Z, & Bethea J.R. (2016). Tumor Necrosis Factor Receptor Associated Factors (TRAFs) 2 and 3 Form a Transcriptional Complex with Phosho-RNA Polymerase II and p65 in CD40 Ligand Activated Neuro2a Cells. Molecular Neurobiology, 54(2), 1301-1313.

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Fluorescence Recovery at Centromeres

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Stable HeLa GFP-Mis18α-expressing cells were grown on glass-bottomed culture dishes (MatTek Corporation). Prior to imaging, growth media were replaced with Leibovitz’s L-15 medium without phenol red (GIBCO) with 10% FBS (Optima, Atlanta Biologicals). Photobleaching was conducted using a Zeiss LSM 510 UV confocal microscope. Two prebleach images were collected. Individual centromeres were bleached with 70 iterations of the 488 nm laser, and the fluorescence recovery at the centromere was assessed at 10 s intervals. Fluorescence recovery at photobleached centromeres was analyzed using ImageJ and normalized to account for sample bleaching (Phair et al., 2004 (link)), and average fluorescence recovery data (GFP-Mis18α, n = 18) was fit to a single exponent curve A × (1 − e^−kt).

Nardi I.K., Zasadzińska E., Stellfox M.E., Knippler C.M, & Foltz D.R. (2016). Licensing of Centromeric Chromatin Assembly through the Mis18α-Mis18β Heterotetramer. Molecular cell, 61(5), 774-787.

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Immunocytochemistry of HEK293 cells

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HEK293 cells were grown overnight on glass coverslips in tissue culture plate (Becton Dickinson and Company, Lincoln Park, NJ). The cells were transfected with pHis-TTP (50 ng DNA/1 mL/well) and incubated overnight. After another 24-h incubation, the cells were used for immunocytochemistry with anti-MBP-hTTP antibodies using a similar procedure as described [7] (link). The slides were examined and imaged with an LSM510 UV confocal microscope (Zeiss, Thornwood, NY).

Cao H., Deterding L.J, & Blackshear P.J. (2014). Identification of a Major Phosphopeptide in Human Tristetraprolin by Phosphopeptide Mapping and Mass Spectrometry. PLoS ONE, 9(7), e100977.

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Immunofluorescence Assay for Embryonic Markers

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Zona pellucidae were removed immediately prior to fixation using acid Tyrode’s solution (137 mM NaCl, 2.68 mM KCl, 1.63 mM CaCl₂, 0.49 mM MgCl₂, 5.55 mM glucose, 0.01 mM polyvinyl pyrrolidone, pH 2.5). Embryos were fixed for 30 min in 2.5% paraformaldehyde in PBS and then permeabilized for 20 min in 0.1% Triton X-100 in PBS. Embryos were then incubated at 4°C overnight in primary antibody (diluted 1:100 for H3K27Ac, H3ac, YAP1, H4ac, c-MYC, total β-catenin, active-β-catenin, AXIN1, and AXIN2; diluted 1:10 for anti-γH2AX; diluted 1:500 for SIRT1), washed, then incubated in Alexa Fluor® 488 anti-rabbit or anti-mouse IgG (Thermo Fisher; 1:500) for 1 h at room temperature, except for anti-γH2AX that is a conjugated antibody. All embryos were mounted in Vectashield containing 1.5 μg/mL DAPI (Vector Laboratories, Burlingame, CA) and slides were scanned using a Zeiss LSM 510 UV confocal microscope. All immunofluorescence experiments were repeated a minimum of three different times using at least 10 embryos per group.

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Investigating Cell Proliferation and Morphology in Kidney Cell Lines

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NRK49f, NRK52e, HK-2, and IMCD-3 cell lines were purchased from ATCC (Mannassas, VA). For proliferation studies, NRK49f cells were seeded in 96-well plates in media containing recombinant GDF11 or TGF-β (R & D Systems) and incubated for 5 d. A MTT assay was used to measure cell viability. For bright-field morphological analysis and preparation of Western blot and PCR extracts, cells were grown in plastic dishes. For αSMA immunohistochemistry and immunofluorescence of SMAD2, E-cadherin, vimentin and αSMA, cells were grown in chamber slides (Nalge Nunc). For immunofluorescence, cells were visualized on a Zeiss LSM510/UV confocal microscope. Inhibitors were added simultaneously with 50 ng/mL GDF11 as follows: 50 ng/mL follistatin (R&D Systems), 500 ng/mL SB-431542 (Sigma), and 10 µM SIS3 (Calbiochem–EMD Millipore, Darmstadt, Germany).

Pons M., Koniaris L.G., Moe S.M., Gutierrez J.C., Esquela-Kerscher A, & Zimmers T.A. (2018). GDF11 induces kidney fibrosis, renal cell epithelial-to-mesenchymal transition and kidney dysfunction and failure. Surgery, 164(2), 262-273.

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Immunofluorescence Staining of Cells

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Indicated cells were plated onto sterile Fisherbrand microscope cover glass slips (Fisher Scientific) and placed in Corning Costar Flat Bottom 6-well Cell Culture Plates (Corning Inc.). The cells were allowed to grow to 80% confluence, washed three times with PBS, and fixed with 4% paraformaldehyde for 20 min. After fixation, the coverslips were transferred to a clean 6-well plate, and cells were again washed with PBS. Cells were then incubated with 0.1% saponin-TBST for 10 minutes at 37°C, after which they were washed three times in 0.1% saponin-TBST. Cells were then blocked for 30 minutes using a background sniper (BIOCARE Medical, Pacheco, CA). Following the blocking step, the cells were washed and incubated with SBP1 primary antibody (MBL) overnight at 1:150 diluted in Diamond Antibody Diluent (Cell Marque, Rocklin CA) in a humid chamber to prevent drying. Cells were then washed three times in 0.1% saponin-TBST. Secondary antibody (Alexafluor-647) was then incubated at 1:200 in Diamond Antibody Diluent for one hour at room temperature in a dark, humid chamber. Cells were then washed three times in 0.1% saponin-TBST, after which they were washed three times in PBS. Cells were mounted using ProLong Gold Antifade reagent with DAPI (Invitrogen). Images were obtained using an LSM510UV confocal microscope (Zeiss).

Elhodaky M., Hong L.K., Kadkol S, & Diamond A.M. (2020). Selenium‐binding protein 1 alters energy metabolism in prostate cancer cells. The Prostate, 80(12), 962-976.

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Visualizing B Cell Nanoparticle Uptake

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Immunosorted (Miltenyi Biotec) and untouched splenic B cells were incubated with Cy5-labeled DEX-NP(OVA-CpG), as indicated; washed with FACS buffer (PBS, 1% FCS, and 0.5 mmol/L EDTA); and transferred onto chamber slides (IBIDI, Martinsried, Germany). Cells were incubated with anti-CD19 antibody (green) and DAPI (blue). The LSM510-UV confocal microscope (Zeiss, Jena, Germany) was used.

Shen L., Tenzer S., Storck W., Hobernik D., Raker V.K., Fischer K., Decker S., Dzionek A., Krauthäuser S., Diken M., Nikolaev A., Maxeiner J., Schuster P., Kappel C., Verschoor A., Schild H., Grabbe S, & Bros M. (2018). Protein corona-mediated targeting of nanocarriers to B cells allows redirection of allergic immune responses. The Journal of allergy and clinical immunology, 142(5).

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Islet Viability Evaluation by Fluorescence

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Islet viability was evaluated with a viability/cytotoxicity assay using calcein AM (Ex 488 nm / Em 515 nm) /ethidium bromide (Ex 488 nm / Em 610 nm) to detect live (green) and dead (red) cells on days 1, 3 and 6 of culture. Fluorescent images were captured under 10 x magnification using a Zeiss LSM 510 UV Confocal Microscope (Carl Zeiss, Germany).

Muthyala S., Safley S., Gordan K., Barber G., Weber C, & Sambanis A. (2016). The effect of hypoxia on free and encapsulated adult porcine islets-an in vitro study. Xenotransplantation, 24(1), 10.1111/xen.12275.

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