Confocal lsm510 microscope

Manufactured by Zeiss

Sourced in United States

The Confocal LSM510 microscope is a high-performance imaging system designed for advanced microscopy applications. It utilizes laser scanning technology to capture detailed, high-resolution images of specimens. The microscope is capable of performing optical sectioning, allowing for the visualization of three-dimensional structures within samples.

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Lab products found in correlation

Axioskop 40 microscope, by Zeiss (2 mentions) Prolong gold mounting medium, by Thermo Fisher Scientific (1 mentions) Paraformaldehyde (pfa), by Thermo Fisher Scientific (1 mentions) Alexa fluor 488 and 594 secondary antibodies, by Thermo Fisher Scientific (1 mentions) Taq dna polymerase, by Takara Bio (1 mentions) Pds 1000 he gene gun, by Bio-Rad (1 mentions) Pgem t easy vector, by Promega (1 mentions) Image pro plus 4, by Media Cybernetics (1 mentions) Anti cleaved caspase 3, by Merck Group (1 mentions) Duolink in situ pla reagents, by Merck Group (1 mentions) Novolink polymer detection kit, by Leica (1 mentions) Axioplan fluorescent microscope, by Zeiss (1 mentions) Duolink in situ proximity ligation assay pla, by Olink (1 mentions) Fluormount g, by Southern Biotech (1 mentions) Anti vegfr2, by Santa Cruz Biotechnology (1 mentions) Mouse anti pecam 1, by Santa Cruz Biotechnology (1 mentions) Axiovision inside4d module, by Zeiss (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)

10 protocols using confocal lsm510 microscope

Immunocytochemistry and Immunofluorescence Protocols

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Immunocytochemistry was performed as described by Kilmartin and Adams (51 (link)). Briefly, yeast were grown to an optical density (OD) equivalent to log phase and were prepared for immunostaining by removing the cell wall. The cell suspension was spotted on polylysine-coated microscope slides, dried, fixed, and permeabilized (10-min incubation in phosphate-buffered saline [PBS] with 0.1% Triton X-100); 1 μg/mL primary antibody (mouse anti-SERCA1a; Abcam) was added and incubated overnight at 4°C, and then 1 μg/mL secondary antibody (Texas Red-tagged goat anti-mouse IgG) was added and incubated for at least 2 h. The slides were stained with 5 μM 3,3′-dihexyloxacarbocyanine [DiOC₆(3)] for 20 min to stain the ER. Antifade mounting medium plus 4′,6-diamidino-2-phenylindole (DAPI) was added before the slides were sealed. Yeast were visualized with a Zeiss confocal LSM 510 microscope.
Immunofluorescence with P. falciparum parasites was performed as described by Pulcini et al. (13 (link)), using the paraformaldehyde/glutaraldehyde fixation method. Parasites were labeled as described by Wang et al. (18 (link)), using 500 nM DHA-biotin probe. Parasites were visualized with a Zeiss confocal LSM 510 microscope.

Moore C.M., Wang J., Lin Q., Ferreira P., Avery M.A., Elokely K., Staines H.M, & Krishna S. (2022). Selective Inhibition of Plasmodium falciparum ATPase 6 by Artemisinins and Identification of New Classes of Inhibitors after Expression in Yeast. Antimicrobial Agents and Chemotherapy, 66(5), e02079-21.

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Visualizing Cytoskeletal and Annexin Dynamics

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Cells were fixed using 4 % paraformaldehyde (Affymetrix, Santa Clara, CA, USA) and stained with rhodamine-conjugated phalloidin, MIEN1 and AnxA2 primary antibodies followed by Alexa Fluor 488 and 594 secondary antibodies (Invitrogen). Cells were mounted on glass coverslips with Prolong Gold mounting medium (Invitrogen). Confocal images were acquired using Zeiss confocal microscope LSM510.

Kpetemey M., Dasgupta S., Rajendiran S., Das S., Gibbs L.D., Shetty P., Gryczynski Z, & Vishwanatha J.K. (2015). MIEN1, a novel interactor of Annexin A2, promotes tumor cell migration by enhancing AnxA2 cell surface expression. Molecular Cancer, 14, 156.

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Cloning and Expression of VpSBP11-GFP Fusion Protein

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The VpSBP11 CDS with XbaI and KpnI sites, but without the termination codon, was amplified using VpSBP11-F3 and R3 (Table 1) from the pGEM-Teasy-VpSBP11 plasmid template with Taq DNA polymerase (TaKaRa Biotechnology). The following cycling program was followed: 94 °C for 3 min, 35 cycles at 94 °C for 30 s, 58 °C for 30 s, 72 °C for 2 min, and extension at 72 °C for 10 min. The amplified products were cloned into the pGEM-Teasy vector (Promega) and transformed into the E. coli strain DH5α. It was then inserted immediately upstream of, and in frame with, the green fluorescent protein (GFP) coding sequence in the pBI221-GFP vector (Clontech Laboratories, Inc., Palo Alto, CA, USA), which had been digested with XbaI and KpnI, to generate pBI221-VpSBP11-GFP. Both the SBP11-containing vector and a background control vector with no insert were delivered into onion epidermal cells using a PDS-1000/He gene gun (Bio-Rad Laboratories Inc., Hercules, CA, USA) at 1100 psi as previously described [43 (link)], and then the cells were cultured in MS media in the dark at 22 °C for 18 h. Following cultivation, GFP accumulation was visualized using a Zeiss confocal microscope (LSM510; Carl Zeiss, Thornwood, NY, USA) with an excitation wavelength of 480 ± 20 nm and an emission wavelength of 510 ± 20 nm.

Hou H., Yan X., Sha T., Yan Q, & Wang X. (2017). The SBP-Box Gene VpSBP11 from Chinese Wild Vitis Is Involved in Floral Transition and Affects Leaf Development. International Journal of Molecular Sciences, 18(7), 1493.

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Immunofluorescence Analysis of Apoptosis and Oxidative Stress

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Fixed midguts and Malpighian tubules from control and LC50-treated individuals were washed and incubated for 24 h at 4 °C in the primary antibody solution (1) anti-cleaved-caspase-3 (cleaved) (1:500) or (2) anti-peroxidase (1:800) (Sigma-Aldrich). Five midguts and five Malpighian tubules were used for anti-cleaved-caspase-3 incubation, and five of each organ were also used for anti-peroxidase incubation. The midguts were washed in PBS followed by incubation in an IgG-FITC secondary antibody, as described above. After washing, the cell nuclei were labeled with TO-PRO-3 (Life Technologies) for 30 min and mounted in Mowiol solution (Fluka). As a negative control, five midguts and five Malpighian tubules were treated as described above, excluding the incubation in the primary antibody (Supp Fig 1). The samples were analyzed and photographed under a Zeiss confocal microscope LSM 510 (Carl Zeiss) in fluorescence mode, at the Nucleus of Microscopy and Microanalysis at the Federal University of Viçosa (NMM/UFV). Fluorescence intensity was quantified using Image-Pro Plus 4.5 software (Media Cybernetics, Silver Spring, USA). For the quantification of the label intensity for the cell death and oxidative stress, six images with a 20X objective (total area = 0.828 mm 2 ) per midgut and Malpighian tubules were arbitrarily selected.

Lopes M.P., Fernandes K.M., Tomé H.V.V., Gonçalves W.G., Miranda F.R., Serrão J.E, & Martins G.F. (2018). Spinosad-mediated effects on the walking ability, midgut, and Malpighian tubules of Africanized honey bee workers. Pest management science, 74(6).

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In Situ Proximity Ligation Assay for Protein Interactions

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An in situ proximity ligation assay (PLA) was performed by using Duolink In Situ PLA reagents (Sigma-Aldrich) to assess the endogenous interaction of ATP7A and VEGFR2 with high specificity and sensitivity. Cells were fixed with 4% paraformaldehyde for 10 min, and then permeabilized in 0.05% Triton X-100 in PBS for 5 min and incubated with primary antibodies against ATP7A (mouse monoclonal, 1:200, Life Span Biosciences, Cat# LS-B8162)) and VEGFR2 (rabbit monoclonal, 1:200, Cell Signaling, Cat# 2479 S) for overnight at 4 °C. After washing, cells were treated with secondary anti-mouse and anti-rabbit antibodies conjugated with oligonucleotides of a PLA probe and then subjected to oligonucleotide hybridization, ligation, amplification, and detection following the manufacturer’s instructions. In this assay, a positive signal is created only when the epitopes of the target proteins are in close proximity (<40 nm). Finally, the signal from each detected pair of PLA probes in the cells with a mounting medium containing DAPI was then counted under Zeiss Confocal LSM 510 microscope (λ_ex 594 nm; λ_em 624 nm). For negative control, cells were treated with single or no primary antibody.

Ash D., Sudhahar V., Youn S.W., Okur M.N., Das A., O’Bryan J.P., McMenamin M., Hou Y., Kaplan J.H., Fukai T, & Ushio-Fukai M. (2021). The P-type ATPase transporter ATP7A promotes angiogenesis by limiting autophagic degradation of VEGFR2. Nature Communications, 12, 3091.

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Brightfield and Confocal Microscopic Imaging

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Brightfield microscopic tissue images were captured on an Axioskop 40 microscope (Carl Zeiss Microimaging, Thornwood, NY). Fluorescent images were captured on a Confocal LSM510 microscope (Carl Zeiss Microimaging).

Shi C., Washington M.K., Chaturvedi R., Drosos Y., Revetta F.L., Weaver C.J., Buzhardt E., Yull F.E., Blackwell T.S., Sosa-Pineda B., Whitehead R.H., Beauchamp R.D., Wilson K.T, & Means A.L. (2014). Fibrogenesis in pancreatic cancer is a dynamic process regulated by macrophage-stellate cell interaction. Laboratory investigation; a journal of technical methods and pathology, 94(4), 409-421.

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Brightfield and Confocal Microscopic Imaging

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Multimodal Microscopy Techniques for Tissue Analysis

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Immunofluorescence was performed as described previously (21 (link)). A Zeiss confocal LSM 510 microscope was used to perform confocal microscopy. Images were processed with ImageJ (http://imagej.net/Coloc_2) to determine Pearson's colocalization coefficients (39 (link)). Epifluorescent microscopy was performed on a Zeiss Axioplan fluorescent microscope. Human thyroid tissue was immunostained using the Novolink polymer detection kit (Leica) as per manufacturer's instructions. Slides were viewed under a light microscope (Zeiss) and images captured using Axiovision software. The Duolink in situ proximity ligation assay (PLA) was performed according to manufacturer's instructions (Olink Bioscience).

Watkins R.J., Imruetaicharoenchoke W., Read M.L., Sharma N., Poole V.L., Gentilin E., Bansal S., Bosseboeuf E., Fletcher R., Nieto H.R., Mallick U., Hackshaw A., Mehanna H., Boelaert K., Smith V.E, & McCabe C.J. (2016). Pro-invasive Effect of Proto-oncogene PBF Is Modulated by an Interaction with Cortactin. The Journal of Clinical Endocrinology and Metabolism, 101(12), 4551-4563.

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Mitochondrial Localization of Fen1 Protein

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Cells were grown in 12-well plates on top of coverslips in the wells and incubated with MitoTracker Red diluted to 0.3 µg/mL in FBS-free media for 30 min. After removing the MitoTracker solution, the samples were incubated for 2 h at room temperature in blocking buffer (0.3% BSA, 0.2% Triton X-100) in PBS (137 mM NaCl, 2.7 mM KCl, 8 mM Na₂HPO₄, and 2 mM KH₂PO₄), and anti-Fen1 (Abcam #70815, diluted 1:700 in blocking buffer) was added and incubated overnight at 4 °C. The cells were washed twice in PBS, then incubated in FITC-labeled anti-rabbit IgG (1:50) for 1 h. The samples were washed twice with PBS, and the coverslips were mounted on slides using Fluormount-G (SouthernBiotech, Birmingham, AL, USA, #0100-01). Imaging was performed on a Zeiss Confocal LSM 510 microscope.

Caston R.A., Fortini P., Chen K., Bauer J., Dogliotti E., Yin Y.W, & Demple B. (2023). Maintenance of Flap Endonucleases for Long-Patch Base Excision DNA Repair in Mouse Muscle and Neuronal Cells Differentiated In Vitro. International Journal of Molecular Sciences, 24(16), 12715.

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Tumor Angiogenesis Immunofluorescence Imaging

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For immunofluorescence analysis paraffin embedded tumor sections were stained with anti-VEGFR2 (Santa Cruz Biotechnologies), antihuman VWF (DakoCytomation) and anti-mouse PECAM1 (Santa Cruz Biotechnologies) antibodies followed by incubation with AlexaFluor 594-conjugated or AlexaFluor 488-conjugated secondary antibodies. Nuclei were counterstained with 4',6-diamidino,2-phenylindole (DAPI, Sigma). Tumor slices were analyzed using Confocal LSM510 microscope equipped with Plan-Neofluar 20 × /0.5 NA and Plan-Apochromat 63 × /1.4 NA oil objectives (Carl Zeiss). Z-stack images were elaborated using AxioVision Inside4D module (Carl Zeiss).

Corsini M., Ravelli C., Grillo E., Dell'Era P., Presta M, & Mitola S. (2021). Simultaneously characterization of tumoral angiogenesis and vasculogenesis in stem cell-derived teratomas. Experimental cell research, 400(2).

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