Lsm 510 confocal

Manufactured by Zeiss

Sourced in Germany

The LSM 510 confocal is a high-performance laser scanning microscope system designed for advanced imaging applications. It provides high-resolution, three-dimensional imaging capabilities with exceptional optical performance. The system utilizes confocal technology to enable optical sectioning and improve image quality by rejecting out-of-focus light.

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25 protocols using lsm 510 confocal

Proximity Ligation Assay for ROCK1-APP and BACE1-APP Interactions

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Proximity ligation assay was performed using Duolink PLA Kit (Sigma‐Aldrich) in 4% PFA‐fixed brain tissue or cells before permeabilization in 0.1% Triton X‐100 for 30 min. The in situ PLA detection kit 596 was used to visualize single‐molecule interactions for ROCK1‐APP or BACE1‐APP. Samples were blocked and incubated with specific primary antibodies at 4°C overnight. Secondary antibodies (anti‐rabbit PLUS and anti‐mouse MINUS probes) were added to the reaction and incubated for 1 hr at 37°C. The hybridized oligonucleotides were ligated for 30 min at 37°C. Then, amplification solution was added with polymerase and incubated for 100 min at 37°C. After mounted with mounting medium, the PL signal was visualized as a chromatic (red) spot and was captured by Confocal LSM 510 (Zeiss).

Hu Y., Ren R., Zhang Y., Huang Y., Cui H., Ma C., Qiu W., Wang H., Cui P., Chen H, & Wang G. (2019). Rho‐associated coiled‐coil kinase 1 activation mediates amyloid precursor protein site‐specific Ser655 phosphorylation and triggers amyloid pathology. Aging Cell, 18(5), e13001.

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Immunofluorescent Staining of ID1 Protein

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Cells were stained with Phalloidin-fluorescein isothiocyanate (1:2, Sigma) and 4′,6-diamidino-2-phenylindole (1:20 000, ThermoFisher) and mounted with Mowiol (Vector). Primary antibody was anti-ID1 (1:500, rabbit monoclonal anti-mouse/human ID1, Biocheck, resp. rabbit polyclonal anti-mouse/human ID1, Abcam), the secondary species-matched antibody was conjugated to AlexaFluor 647 (1:500, anti-rabbit IgG Invitrogen). Images were acquired with confocal microscopy (Zeiss confocal LSM 510).

Koch M.S., Czemmel S., Lennartz F., Beyeler S., Rajaraman S., Przystal J.M., Govindarajan P., Canjuga D., Neumann M., Rizzu P., Zwirner S., Hoetker M.S., Zender L., Walter B., Tatagiba M., Raineteau O., Heutink P., Nahnsen S, & Tabatabai G. (2020). Experimental glioma with high bHLH expression harbor increased replicative stress and are sensitive toward ATR inhibition. Neuro-oncology Advances, 2(1), vdaa115.

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Proximity Ligation Assay for STIM1-TRPC1 Interaction

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Freshly isolated VSMCs were studied using the Duolink in situ proximity ligation assay (PLA) detection kit 563 (Olink, Uppsala, Sweden) (Soderberg et al. 2008). Cells were adhered to coverslips, fixed in PBS containing 4% paraformaldehyde for 15 min, and permeabilized in PBS containing 0.1% Triton X‐100 for 15 min. Cells were blocked for 1 h at 37°C in blocking solution, and incubated overnight at 4°C with anti‐STIM1 and anti‐TRPC1 antibodies (both at dilution 1:200) in antibody diluent solution. Cells were labelled with combinations of either anti‐goat PLUS/anti‐rabbit MINUS or anti‐goat PLUS/anti‐mouse MINUS depending on animal species used for 1 h at 37°C. Hybridized oligonucleotides were ligated for 30 min at 37°C prior to amplification for 100 min at 37°C. Red fluorescence‐labelled oligonucleotides were then hybridized to rolling circle amplification products, and visualized using a Confocal LSM 510 (Carl Zeiss).

Shi J., Miralles F., Birnbaumer L., Large W.A, & Albert A.P. (2016). Store‐operated interactions between plasmalemmal STIM1 and TRPC1 proteins stimulate PLCβ1 to induce TRPC1 channel activation in vascular smooth muscle cells. The Journal of Physiology, 595(4), 1039-1058.

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Proximity Ligation Assay for TRPV4 Localization

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Freshly isolated ECs were studied using the Duolink in situ proximity ligation assay (PLA) detection kit 563 (Olink, Uppsala, Sweden) [50] (link). Cells were plated onto coverslips, fixed with PBS containing 4% paraformaldehyde for 15 min, and permeabilized in PBS containing 0.1% Triton X-100 for 15 min. Cells were blocked for 1 h at 37 °C in blocking solution, and incubated overnight at 4 °C with anti-TRPV4 and T1E3 antibodies (both at dilution 1:200) in antibody diluent solution. Cells were labelled with combinations of either anti-goat PLUS/anti-rabbit MINUS 1 h at 37 °C. Hybridized oligonucleotides were ligated for 30 min at 37 °C prior to amplification for 100 min at 37 °C. Red fluorescence-labelled oligonucleotides were then hybridized to rolling circle amplification products, and visualized using a Confocal LSM 510 (Carl Zeiss).

Greenberg H.Z., Carlton-Carew S.R., Khan D.M., Zargaran A.K., Jahan K.S., Vanessa Ho W.S, & Albert A.P. (2017). Heteromeric TRPV4/TRPC1 channels mediate calcium-sensing receptor-induced nitric oxide production and vasorelaxation in rabbit mesenteric arteries. Vascular Pharmacology, 96-98, 53-62.

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Characterization of Blue-Emitting Quantum Dots

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UV–vis spectra were obtained
from the UV–vis spectrometer of Hewlett-Packard. PL spectra
were obtained from L550B luminescence spectrometer of Perkin-Elmer.
TEM and HRTEM images were recorded with FEI Tecnai T20 at an accelerating
voltage of 200 kV. AFM topographic images were obtained from XE-100
model of PSIA. FTIR spectra were obtained from Nicolet IR 200. XPS
data were obtained from Multilab 2000 of Thermo Scientific. Raman
spectra were obtained from LabRAM high-resolution dispersive Raman
microscope of Horiba Jobin Yvon using a 633 nm He–Ne laser
beam. The NMR spectra were recorded using an Avance digital 400 MHz
Bruker Spectrometer (Germany). ζ Potential measurements were
made with a Zetasizer ELS Z (Otsuka Electronics, South Korea). Confocal
fluorescent images were taken with a confocal laser microscopy system
(Confocal, LSM510, Carl Zeiss, Oberkochen 73447, Germany). The measurement
of QY of BPQDs and PEG-BPQDs was attempted by the reference point
method typically attempted for estimating QYs of other blue-emitting
fluorescent (nano)materials by a comparison with quinine sulfate (QY
of 0.54 in 0.1 M H₂SO₄).

Lee M., Park Y.H., Kang E.B., Chae A., Choi Y., Jo S., Kim Y.J., Park S.J., Min B., An T.K., Lee J., In S.I., Kim S.Y., Park S.Y, & In I. (2017). Highly Efficient Visible Blue-Emitting Black Phosphorus Quantum Dot: Mussel-Inspired Surface Functionalization for Bioapplications. ACS Omega, 2(10), 7096-7105.

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Visualization of Cilia and Sperm Structures

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Cells grown on glass coverslips were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X‑100 in PBS, incubated in blocking buffer (2% heat‐inactivated goat serum, 0.2% Triton X‑100 in PBS) and probed with primary antibodies diluted in blocking buffer. After washing, appropriate secondary antibodies were added along with DAPI, before mounting in Mowiol. Cilia formation was induced by serum starvation for 24–48 h and visualized by anti‐acetylated tubulin, anti‐Arl13b or anti‐gamma‐tubulin antibodies. In some cases, cells were pre‐treated with different drugs for 10 h before staining. For analysis of the sperm, caudal epididymides from sexually mature males were finely minced and the sperm released into the buffer was collected and mounted. The slides were then air‐dried, fixed in 1.5% paraformaldehyde (v/v in PBS), permeabilized using 0.2% Triton X‐100, blocked in 2% BSA and stained with primary antibodies and DAPI. Fluorescence microscopy was performed using Zeiss Axioplan 2 Upright or Zeiss Confocal LSM 510 and analysed by using ImageJ software (https://imagej.nih.gov/ij/index.html).

Kim Y., Osborn D.P., Lee J., Araki M., Araki K., Mohun T., Känsäkoski J., Brandstack N., Kim H., Miralles F., Kim C., Brown N.A., Kim H., Martinez‐Barbera J.P., Ataliotis P., Raivio T., Layman L.C, & Kim S. (2017). WDR11‐mediated Hedgehog signalling defects underlie a new ciliopathy related to Kallmann syndrome. EMBO Reports, 19(2), 269-289.

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Impression Cytology for HLA-DR Expression

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IC was performed to evaluate the HLA-DR expression at the temporal bulbar conjunctiva, and was conducted 24 hours after the IVCM assessment to avoid misinterpretation due to the technical execution of the confocal microscopy. Samples were collected using Millicell-CM 0.4 µm (Millipore, Bedford, MA); the specimen acquiring procedure was performed as previously described.¹⁸ (link)^,¹⁹ (link) Zeiss Confocal LSM 510 (Carl Zeiss MicroImaging GmbH, Vertrieb, Germany) was used to visualize cells. Five different fields for each sample were evaluated. Positive (red nucleus and green cytoplasm) and negative (red nucleus) cells were counted. Minor irregularities that may have been present after cutting the membranes were normalized by reporting the percentage of positive cells for HLA-DR.
All evaluations of impression cytology samples were performed by two independent observers (authors L.B. and R.D.A.). IVCM (author M.L.) and IC (authors L.B. and R.D.A.) operators were masked for grouping and subject history of patients. Both eyes for patients were evaluated in the study, but one eye per subject was randomly chosen (using a computer-generated random number list) for the statistical analysis.

Mastropasqua L., Lanzini M., Brescia L., D'Aloisio R., Nubile M., Ciancaglini M., D'Amario C., Agnifili L, & Mastropasqua R. (2021). Face Mask-Related Ocular Surface Modifications During COVID-19 Pandemic: A Clinical, In Vivo Confocal Microscopy, and Immune-Cytology Study. Translational Vision Science & Technology, 10(3), 22.

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Insulin Immunostaining in Islet Cells

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Confocal microscopy was performed as described previously (Aggarwal et al., 2011 (link)). NIT-1 cells and Mouse islets were plated on cover slips, fixed with 2 % formaldehyde, washed with PBS, permeabilized with 0.1% Trion–X100 and incubated with 2 % goat serum for one hour. The cells were then incubated with a primary antibody specific for Insulin (Cell Signaling Technology, Cambridge, MA) overnight at 4°C, then washed with PBS and incubated with a secondary antibody conjugated with Alexa Fluor 594 (Molecular Probes, Eugene, OR) for one hour followed by three washings with PBS. Finally, cells were mounted with mounting medium from Molecular Probes (Carlsbad, CA) and images of insulin immunostained cells were acquired under an oil immersion objective (x40) with a confocal laser microscope (Zeiss Confocal LSM510, Carl Zeiss MicroImaging Inc.Thornwood, NY, USA) equipped with an argon-krypton laser.

Aggarwal S., Shailendra G., Ribnicky D.M., Burk D., Karki N, & Wang Q. (2015). An Extract of Artemisia dracunculus L. stimulates insulin secretion from β cells, activates AMPK and suppresses inflammation. Journal of ethnopharmacology, 170, 98-105.

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Immunofluorescence Analysis of Aortic Plaque

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For Immunofluorescence, 6 μm frozen sections of aortic roots were fixed and permeabilized with cold-acetone for 5 min and blocked in PBS containing 3% BSA for 1 hour at room temperature. Sections were stained with rat anti-CD31 (BD Pharmingen, 550274, 1:30), rabbit anti-p16 (1:100, ab51243, Abcam), rabbit anti-p21 (1:400, 2947, Cell Signaling), rabbit anti-γH2AX (2577, 1:800, Cell Signaling), cleaved caspase3 (1:400, 9661, Cell Signaling), Importin-α3 (1:100, ab84706, Abcam) and rabbit anti-MEKK3 (Cell Signaling) in PBS at 4 oC overnight. After three washes with PBS, the sections were further incubated with Alexa Flour 488-conjugated donkey anti-Rat IgG (Invitrogen, A21208, 1:300) and Alexa Flour 555-conjugated goat anti Rabbit IgG (Invitrogen, 1:300) in PBS for 1 hour at room temperature. Nuclei were stained for 5min at room temperature in PBS containing DAPI (Cell signaling, 4083, 0.5μg/ml). Coverslips were mounted with ProLong Gold antifade reagent (Invitrogen). Images were acquired on an upright Carl Zeiss LSM 510 confocal. The data were calculated from at least 40 cells for each group with 6–10 mice. For each mouse, 2–5 plaques were selected and used for quantification. Tunnel protocol was performed as described by manufacturer’s protocol (Millipore, ApopTaq Peroxidase Detection kit).

Zhou H., Yang D., Cheng H.S., McCoy M.G., Pérez-Cremades D., Haemmig S., Wong D., Chen L, & Feinberg M.W. (2022). miR-181b regulates vascular endothelial aging by modulating a MAP3K3 signaling pathway. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 36(6), e22353.

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BrdU Incorporation Assay

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Cells were incubated with 10 μM BrdU (Sigma) for 1 hr 30 min. Cells were immediately fixed with 4% PFA and incubated with 2N HCl to denature DNA to facilitate antibody penetration. 0.1mM sodium borate buffer was used to neutralize HCl prior to routine ICC. Cells were imaged on a Zeiss LSM 510 confocal. 5 random fields of view were taken for each individual experiment to sample 200–300 cells.

Sarraf S.A., Sideris D.P., Giagtzoglou N., Ni L., Kankel M.W., Sen A., Bochicchio L.E., Huang C.H., Nussenzweig S.C., Worley S.H., Morton P.D., Artavanis-Tsakonas S., Youle R.J, & Pickrell A.M. (2019). PINK1/Parkin Influences Cell Cycle by Sequestering TBK1 at Damaged Mitochondria, Inhibiting Mitosis. Cell reports, 29(1), 225-235.e5.

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