Lsm 510 confocal microscope

Manufactured by Zeiss

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The LSM 510 confocal microscope is a high-resolution imaging system designed for advanced microscopy applications. It utilizes laser scanning technology to capture detailed, three-dimensional images of samples with high contrast and resolution.

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LSM 510 (2630 citations) Confocal microscope (860 citations) LSM 510 microscope (264 citations) Zeiss 510 confocal microscope (104 citations) Confocal LSM 510 (10 citations) Carl Zeiss LSM 510 META confocal microscope (4 citations) LSM 510 duo vario confocal microscope (2 citations) LSM 510 or 800 confocal microscope (2 citations) LSM 510 laser scan confocal microscope (2 citations) B29/Zeiss LSM 510 META laser confocal microscope (2 citations)

1 169 protocols using lsm 510 confocal microscope

Transient Expression of TaRac6 Fusion Proteins

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GV3101 carrying pCAMBIA-1302-TaRac6-GFP^C, pCAMBIA-1302-GFP, pBinGFP2-GFP^N-TaRac6 or 35S-mCherry plasmids were cultured in LB (50 μg/mL kanamycin and 50 μg/mL rifampicin) for 1–2 days. The cells were collected and suspended as described by Zhao et al. (2018) (link). GFP, GFP^N-TaRac6, TaRac6-GFP^C of A. tumefaciens were co-injected into leaves of tobacco plants 4–6 weeks old with mCherry, respectively. Two days later plant tissue samples were harvested. The GFP images were taken under a LSM510 Confocal Microscope (Zeiss, Germany) with 488 nm laser lines. The mCherry images were taken under a LSM510 Confocal Microscope (Zeiss, Germany) with 584 nm laser lines.
The expression of TaRac6-GFP^C and GFP^N-TaRac6 were further confirmed by western blot. The total proteins of injected tobacco leaves were extracted using the Native lysis buffer (Solarbio, Beijing, China). Specifically, 10 μL PMSF (100 mM) and 10 μL protease inhibitor cocktail (EDTA-Free, 100 × in DMSO) were added per ml of lysate. The extraction of total protein and the western blot procedure used are described in Zhao et al. (2018) (link).

Zhang Q., Zhang X., Zhuang R., Wei Z., Shu W., Wang X, & Kang Z. (2020). TaRac6 Is a Potential Susceptibility Factor by Regulating the ROS Burst Negatively in the Wheat–Puccinia striiformis f. sp. tritici Interaction. Frontiers in Plant Science, 11, 716.

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Immunofluorescence Staining for YAP and Cholesterol

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Cells were fixed in PBS 100 mM sucrose for 30 min, permeabilized with PBS 0.5% Triton X-100 for 3 min before incubation with anti-YAP antibody 1/100 in PBS for 2 h at room temperature. Cells were washed three times and incubated with fluorescently-labeled phalloidin PBS supplemented with DAPI for 1 h. Cells were washed three times before incubation with fluorescently labeled secondary antibody 1/250 in PBS for 1 h. Hydrogels were mounted in mowiol and imaged on a Zeiss LSM510 confocal microscope.
Cholesterol staining was performed according to manufacturer’s protocol using the Cholesterol Cell-Based Detection Assay Kit (10009779) from Cayman Chemicals (Ann Arbor, Michigan, USA). Cells were permeabilized with PBS 0.5% Triton X-100 for 3 min before incubation with anti-GEF-H1 antibody (B4/7) for 1 h. Cells were washed three times before incubation with fluorescently labeled secondary antibody 1/250 in PBS for 1 h. Coverslips were mounted in mowiol and imaged on a Zeiss LSM510 confocal microscope.

Boulter E., Estrach S., Tissot F.S., Hennrich M.L., Tosello L., Cailleteau L., de la Ballina L.R., Pisano S., Gavin A.C, & Féral C.C. (2018). Cell metabolism regulates integrin mechanosensing via an SLC3A2-dependent sphingolipid biosynthesis pathway. Nature Communications, 9, 4862.

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Visualizing Erythrocytes and Giant Membrane Vesicles

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For erythrocytes, 4 million cells were added to 200 µL per well of an 8-well LabTek chamber (Nunc). Calcein was added at 20 µg/mL and BODIPY-MUS:OT 2:1 amph-AuNPs were added at 0.28 mg/mL. Treatment medium was prepared before the addition of cells and samples were rocked back and forth gently without mixing or agitation with a pipette. Samples were imaged with a LSM 510 confocal microscope (Carl Zeiss). Z-stacks were collected using an optical slice thickness of 1 µm, and images were analyzed with Zeiss LSM software.
For GMVs, confocal samples were prepared in 8-well LabTek chambers (Nunc) and, prior to adding GMVs, we first prepared solutions of 50 mM glucose in water with BODIPY-MUS:OT 2:1 amph-AuNPs at 0.07 mg/mL. GMV harvests in sucrose were mixed 1–2x with a pipette and added at a 1:4 ratio in a sample well. Notably, samples were only rocked back and forth after preparation and there was no additional mixing of GMVs with AuNPs in the well. Samples were incubated 1–3 hr at 22°C, unless otherwise noted. Samples were then imaged with a LSM 510 confocal microscope (Carl Zeiss). Z-stacks were collected using an optical slice thickness of 1 µm, and images were analyzed with Zeiss LSM software.

Atukorale P.U., Yang Y.S., Bekdemir A., Carney R.P., Silva P.J., Watson N., Stellacci F, & Irvine D.J. (2015). Influence of the Glycocalyx and Plasma Membrane Composition on Amphiphilic Gold Nanoparticle Association with Erythrocytes. Nanoscale, 7(26), 11420-11432.

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Fluorescence Microscopy for Visualizing GFP and AmCyan Constructs

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U2OS cells transiently transfected with GFP- and AmCyan-tagged expression constructs or treated with commercial Baculovirus reagents were fixed on coverslips with 3% PFA for 20 min, washed with 0.1 M glycine in PBS and stained with Hoechst solution or propidium iodide for 5 min at room temperature. For immunofluorescence, after fixation with PFA, the cells were permeabilized with 0.1% Triton-X100 in PBS for 5 min, and blocked in PBS containing 5% BSA for 30 min at room temperature; then the slides were incubated with the appropriate antibodies (1:100) for 2 h at 37°C. Following 3 washings with PBS, samples were stained with secondary antibodies for 1 h at room temperature. Images were acquired with LSM510 confocal microscope (Zeiss), and processed using ImageJ software, freely available on the net. Imaging of live U2OS cells, grown on Nunc Glass Base dishes (Thermo Fisher Scientific) was performed on LSM510 confocal microscope (Zeiss), equipped with a cells incubator. Images were acquired every 2 min.

Marcassa E., Raimondi M., Anwar T., Eskelinen E.L., Myers M.P., Triolo G., Schneider C, & Demarchi F. (2017). Calpain mobilizes Atg9/Bif-1 vesicles from Golgi stacks upon autophagy induction by thapsigargin. Biology Open, 6(5), 551-562.

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Immunocytochemistry and Cell Death Assays

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Immunocytochemistry assays were performed in 8-well slide chambers. Briefly, cells were fixed in 4% paraformaldehyde (PFA) for 20 minutes, permeabilized with Triton X-100 0.1% in PBS, and non-specific epitopes were blocked in 10% bovine serum albumin (BSA) in 1 × PBS for 1 hour at room temperature. Immunostaining was accomplished by incubating primary antibodies overnight at 4 °C. Samples were incubated for 2 hours at room temperature with Alexa Fluor 555 conjugated secondary antibodies diluted at 1 in 250 (MeridianLife Science Inc., Memphis, TN), and nuclei were stained with Hoechst (2 μM Hoechst33258). Pictures were taken with a Zeiss LSM 510 confocal microscope and a Zeiss Axioplan-2 deconvolution microscope.
To assess cell death, primary human RPE cells and ARPE-19 cells were fixed with methanol for 15 minutes, washed with 1 × PBS, then loaded with 2 μM Hoechst dissolved in a Locke’s solution (Promega) and incubated for another 15 minutes before imaging. Cells were then viewed by using a Zeiss LSM 510 confocal microscope under UV fluorescence. Images were recorded, and cell apoptosis was assessed by using an automated unbiased method^{70 (link)}.

Jun B., Mukherjee P.K., Asatryan A., Kautzmann M.A., Heap J., Gordon W.C., Bhattacharjee S., Yang R., Petasis N.A, & Bazan N.G. (2017). Elovanoids are novel cell-specific lipid mediators necessary for neuroprotective signaling for photoreceptor cell integrity. Scientific Reports, 7, 5279.

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Transient Transformation of Petunia Protoplasts

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Protoplasts were isolated from petals of wild-type (M1xV30), rab5a X2043 , or ph4 mutants and transiently transformed with plasmids of interest, respectively. 24 hours after transformation, transformed cells were treated with 16.5 mM (final concentration) wortmannin (LC Laboratoriesâ) for 30 mins in the dark. After the treatment, protoplasts were centrifuged, resuspended and kept in fresh buffer for a variable period before imaging with a Zeiss LSM510 confocal microscope using a 40x/1.2 water objective.
Yeast two hybrid and bimolecular fluorescence complementation (BiFC) assay Yeast two-hybrid assays were performed as reported before (Quattrocchio et al., 2006) . In BiFC assays, plasmids encoding N-terminal fusions of protein of interest to nYFP and cYFP and, to mark the plasma membrane of transformed cells, RFP-SYP122 were transformed into protoplasts isolated from petunia M1xV30 (wild-type) petals. Transfected cells were imaged with a Zeiss LSM510 confocal microscope with a 40x/1.2 water objective at 24 hours after transformation. Over hundred transfected cells expressing RFP-SYP122 were observed for each independent experiment, and around 20 cells were imaged by confocal microscopy.

Li S., Cerri M., Strazzer P., Li Y., Spelt C., Bliek M., Vandenbussche M., Martínez-Calvó E., Lai B., Reale L., Koes R, & Quattrocchio F.M. (2021). An ancient RAB5 governs the formation of additional vacuoles and cell shape in petunia petals. Cell reports, 36(13).

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Duolink™ Protein Interaction Analysis

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Duolink™ (OLINK Bioscience) was performed according to the manufacturer’s instructions. Briefly, the cells were incubated for 30 min on IBItreat μ-channels (IBIDI) coated with poly-L-Lysine, fixed for 10 min in 4% paraformaldehyde prewarmed at 37 °C, permeabilized in PBS, 0.2% saponin for 10 min and blocked with 0.2% BSA in PBS, 0.1% saponin. Primary antibodies used were: mouse anti-CD3ε (OKT3, Biolegend), mouse anti-CD3ζ (6B.10.2, Santa Cruz Biotechnology), rabbit anti-IRAP (a generous gift from S. Keller, Virginia University, USA), mouse anti-Lck (3A5, Santa Cruz Biotechnology), rabbit anti-LAT (Millipore), rabbit anti-Rab7 (H-50, Santa Cruz Biotechnology) and rabbit anti-IRAP (D7C5), mouse anti-IRAP (3E1) and rabbit anti-ZAP-70 (D1C10E) (all from Cell Signaling Technology). All primary antibodies were diluted at 1/100, except mouse anti-CD3ζ (1/50). After washing the cells, PLA probes were added, followed by hybridisation, ligation, and amplification for 100 min at 37 °C. Protein interactions were visualised after incubation with the detection solution. Fluorescence signal was acquired on an LSM 510 Zeiss confocal microscope.

Evnouchidou I., Chappert P., Benadda S., Zucchetti A., Weimershaus M., Bens M., Caillens V., Koumantou D., Lotersztajn S., van Endert P., Davoust J., Guermonprez P., Hivroz C., Gross D.A, & Saveanu L. (2020). IRAP-dependent endosomal T cell receptor signalling is essential for T cell responses. Nature Communications, 11, 2779.

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Live Imaging of Bacterial Colony Growth

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A mixed colony was picked into a well of a 96-well plate and grown overnight in 100 μl of 2XYT media. 1 μl was taken out and diluted into 1 ml of 2xYT media. 1 μl was again taken out of this dilution and and diluted into 1 ml of 2xYT media. 300 μl of it was dispensed on a agarose plate. The next day, a tip was dipped in a colony, then swirled in 1 ml of 2xYT media. 10 μl -20 μl of this media was placed in the center of a small agarose plate. Small plates were made of a 4.5% agarose main bottom layer topped with a superficial layer of 3% agarose. The plates were placed on a heated microscope stage at 33-35C and cover-slipped leaving an air interface. Images were taken with 20X/0.5 Plan-Neofluar Zeiss lens on a LSM 510 Zeiss Confocal Microscope at 4 × optical zoom. The developing colony was manually refocused every 20 min by moving the stage up in the Z position by about 20 μm.

Tomoiaga D., Bubnell J., Herndon L, & Feinstein P. (2022). High rates of plasmid cotransformation in E. coli overturn the clonality myth and reveal colony development. Scientific Reports, 12, 11515.

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Investigating Prostate Cancer Cell Lines

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COS7 cells and the human prostate cancer cell lines C4-2B/Rx2^dox, 22Rv1/Rx2^dox and LNCaP/Rx2^dox were previously described (8 (link), 17 (link)) and have been passaged for less than 6 months. PCa cells were maintained in RPMI-1640 supplemented with 10% FBS and COS7 cells were maintained in DMEM with 5% FBS. Hygromycin (50μg/ml) and puromycin (1μg/ml) were used to select cells that had incorporated the Rx2^dox and the shSNAI2 lentiviral vectors, respectively. Two days before initiation of hormone treatment, 10% FBS was replaced with 5% CSS, and all experiments were performed in the absence of any selection marker. AR and RUNX2 immunofluorescence was performed using the N20 and M70 primary antibodies and fluorescein- and rhodamine-conjugated secondary antibodies respectively. Cells were mounted using Vectashield mounting medium with DAPI (Vector Laboratories Inc., Burlingame, CA) and viewed using an LSM 510 Zeiss confocal microscope (Carl Zeiss, Thornwood, NY). Fluorescence recovery after photobleaching (FRAP) was carried out as previously described (8 (link)).

Little G.H., Baniwal S.K., Adisetiyo H., Groshen S., Chimge N.O., Kim S.Y., Khalid O., Hawes D., Jones J.O., Pinski J., Schones D.E, & Frenkel B. (2014). Differential effects of RUNX2 on the androgen receptor in prostate cancer: synergistic stimulation of a gene set exemplified by SNAI2 and subsequent invasiveness. Cancer research, 74(10), 2857-2868.

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Immunofluorescence Microscopy Assay

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Cells were plated in 6-well plate with sterilized glass coverslips. After the indicated treatments or transfections, cells were fixed with 3.7% formaldehyde in DMEM at 37 °C for 15 min, and then washed three times with ice-cold PBS. Subsequently, cells on coverslips were permeabilized by 0.2% Triton X-100 at RT for 15 min, blocked by 5% fetal bovine serum, then incubated with indicated primary and corresponding secondary conjugated antibodies at room temperature for 120 min and 60 min, respectively. Images were captured with a LSM 510 Zeiss confocal microscope.

Wu H., Wei H., Zhang D., Sehgal S.A., Zhang D., Wang X., Qin Y., Liu L, & Chen Q. (2020). Defective mitochondrial ISCs biogenesis switches on IRP1 to fine tune selective mitophagy. Redox Biology, 36, 101661.

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