Leica sp5 confocal microscope

Manufactured by Leica camera

Sourced in Germany, United States, Israel

The Leica SP5 is a high-performance confocal microscope designed for advanced imaging applications. It features a modular design, allowing for customization to suit specific research needs. The Leica SP5 provides high-resolution, multi-dimensional imaging capabilities, enabling detailed analysis of biological samples.

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Lab products found in correlation

Vectashield, by Vector Laboratories (7 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (6 mentions) Cytofix, by BD (4 mentions) Perm wash, by BD (4 mentions) Leica las af software, by Leica camera (4 mentions) Leica application suite software, by Leica camera (4 mentions) Alexa fluor 488, by Thermo Fisher Scientific (4 mentions) Leica sp5 microscope, by Leica camera (3 mentions) Fitc phalloidin, by Merck Group (3 mentions) Sp5 confocal microscope, by Leica camera (3 mentions) In situ cell death detection kit, by Roche (3 mentions) Bovine serum albumin (bsa), by Merck Group (3 mentions) Hoechst, by Thermo Fisher Scientific (3 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions) Prolong gold antifade reagent, by Thermo Fisher Scientific (3 mentions) Leica dmi6000b microscope, by Leica camera (2 mentions) Las af software, by Leica Microsystems (2 mentions) Prolong gold antifade, by Thermo Fisher Scientific (2 mentions) Leica stereomicroscope, by Leica camera (2 mentions) Phalloidin, by Thermo Fisher Scientific (2 mentions)

240 protocols using leica sp5 confocal microscope

Visualizing ORF-GFP Fusion Proteins in Breast Cancer Cells

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A series of ORF-GFP fusion plasmids were transfected into MDA-MB-231 cells, and GFP fluorescence was directly visualized by Nikon Eclipse Ti-U fluorescence microscope. MDA-MB-231 and MCF7 cells were plated on glass coverslips, fixed with 4% paraformaldehyde at room temperature for 15 min, rinsed three times with phosphate-buffered saline Triton (PBSTx; 0.3% Triton X-100), incubated with anti-ASRPS antibody and anti-FLAG antibody overnight at 4°C, washed twice with Tris-buffered saline Triton (TBSTx), and incubated with FITC secondary IgG antibodies (Beyotime) at room temperature for 1 h in the dark. Cell nuclei were stained with DAPI. Fluorescence images were analyzed using Leica SP5 Confocal Microscopes.
Tumor tissues were fixed and frozen in optimum cutting temperature compound (Sakura). Sections of 10-µm thickness were cut at −20°C. The sections were incubated with CD31 overnight at 4°C. After three rinses with PBST (0.3% Triton X-100), sections were incubated for 1 h at room temperature with Cy3 secondary IgG antibodies in the dark (Beyotime). Cell nuclei were stained with DAPI. Fluorescence images were analyzed using Leica SP5 Confocal Microscopes.

Wang Y., Wu S., Zhu X., Zhang L., Deng J., Li F., Guo B., Zhang S., Wu R., Zhang Z., Wang K., Lu J, & Zhou Y. (2019). LncRNA-encoded polypeptide ASRPS inhibits triple-negative breast cancer angiogenesis. The Journal of Experimental Medicine, 217(3), jem.20190950.

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Visualizing Inflammasomes and Membrane Markers in BMDMs

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For visualization of inflammasomes, untreated or treated BMDMs were washed three times with PBS and fixed with 4% paraformaldehyde at room temperature for 15 min, followed by blocking in 10% normal goat serum (005000121, Jackson ImmunoResearch) supplemented with 0.1% saponin (47036, Sigma) for 1 hr. Cells were incubated with a rabbit anti-ASC antibody (1:500 dilution, clone AL177, AG-25B-0006-C100, AdipoGen) overnight at 4 °C. An anti-rabbit secondary Rhodamine red antibody (111295144, Jackson ImmunoResearch) was used. Cells were counterstained in DAPI mounting medium (H-1200, Vecta Labs). Inflammasome specks and BMDMs were visualized, counted, and imaged using a Leica SP5 confocal microscope. For visualization of HBL and cell membrane, BMDMs were stimulated with HBL component B (5 μg/ml) for 1 hr, washed three times with PBS and fixed with 4% paraformaldehyde at room temperature for 15 min, followed by blocking in 1% BSA in PBS for 1 hr. Cells were incubated with a mouse anti-HBL-B antibody (1:200 dilution in 1% BSA)⁴⁰ and a rat FITC-conjugated anti-CD11b antibody (1:200 dilution in 1% BSA, 101205, BioLegend) overnight at 4 °C. PBS containing 0.05% Tween-20 was used to wash between incubation steps. An anti-mouse secondary Rhodamine red antibody (115295146, Jackson ImmunoResearch) was used. BMDMs were analysed using a Leica SP5 confocal microscope.

Mathur A., Feng S., Hayward J.A., Ngo C., Fox D., Atmosukarto I.I., Price J.D., Schauer K., Märtlbauer E., Robertson A.A., Burgio G., Fox E.M., Leppla S.H., Kaakoush N.O, & Man S.M. (2018). A multi-component toxin from Bacillus cereus incites inflammation and shapes host outcome via the NLRP3 inflammasome. Nature microbiology, 4(2), 362-374.

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Visualizing Bacterial, ROS, and Oil Bodies in Seeds

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Bacteria, ROS and OBs inside the seeds were visualized by CLSM. Seeds were collected at the specified hours after treatment and were transversally cut with a scalpel. A drop of glycerol was applied to the sections, which were placed into 1.5-mm-thick cover glasses (22 × 22 mm). The bacteria were stained during inoculum preparation by CellTracker CM-DiI dye or in situ by Hoechst solution. Oil bodies and ROS were stained with Nile red (1:1,000 v/v) or dihydrorhodamine 123 (1:500 v/v), respectively. The images were acquired using a Leica SP5 confocal microscope with a ×HCX IRAPO L 25.0×0.95 WATER objective. To image the purified OB suspension, a drop of Nile red and Fast Green FCF-stained preparation was applied onto a patch of polymerized 1% agarose on a glass slide, which was covered with a coverslip. The images were acquired using a Leica SP5 confocal microscope with HCX PL APO lambda blue 63.0×1.40 OIL UV objective. Image processing was performed using Leica Application Suite Advance Fluorescence v.2.7.3.9723 (LCS Lite, Leica Microsystems) and FIJI/ImageJ software. Laser settings, scan speed, photomultiplier detector gain and pinhole aperture were constant for all acquired image stacks for each experiment.

Berlanga-Clavero M.V., Molina-Santiago C., Caraballo-Rodríguez A.M., Petras D., Díaz-Martínez L., Pérez-García A., de Vicente A., Carrión V.J., Dorrestein P.C, & Romero D. (2022). Bacillus subtilis biofilm matrix components target seed oil bodies to promote growth and anti-fungal resistance in melon. Nature Microbiology, 7(7), 1001-1015.

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Actin Cytoskeleton Visualization in Cells

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Cells were grown from 6 h to overnight at 36°C, fixed with methanol at -20°C, mounted with Mowiol (Calbiochem), and cell imaging was performed under a Leica SP5 Confocal Microscope. For actin staining, cells were fixed with formaldehyde 60%, washed twice in PM Buffer (35 mM K-Phos pH 6.8, 0.5 mM MgSO₄), permeabilized with 1% Triton X-100, washed twice in PM Buffer, and stained with phalloidin conjugated-Alexa Fluor 488 (Invitrogen, Molecular probes) for 40 min in the dark. Cells were mounted and cell imaging was performed under a Leica SP5 Confocal Microscope. Image analysis and measurements were carried out using Image J.

Gómez-Hierro A., Lambea E., Giménez-Zaragoza D., López-Avilés S., Yance-Chávez T., Montserrat M., Pujol M.J., Bachs O, & Aligue R. (2015). Ssp1 CaMKK: A Sensor of Actin Polarization That Controls Mitotic Commitment through Srk1 in Schizosaccharomyces pombe. PLoS ONE, 10(11), e0143037.

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3D Coculture Spheroid Analysis

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Microscopy pictures were taken using either an inverted brightfield Olympus IX73 or a Leica SP5 confocal microscope. Image analysis was performed with Fiji (ImageJ, NIH) or Volocity® 3D Image Analysis Software (v 6.3, Perkin Elmer, Hamburg, Germany). The measurement of spheroid size was conducted using brightfield microscope images by drawing a line across the center of the spheroid. Only cells/spheroids in focus for each image were measured. In 3D–3D coculture experiments, analysis of interactions between tumor spheroids and HUVECs in the hydrogel construct was performed. Therefore, contact points of MCF-7 and HUVECs were classified as (1) internal contact, (2) external contact, and (3) no contact. MCF-7 cells were easily identified based on their uniform spherical shape and HUVECs were stained for CD31. Analysis was performed directly using the Leica SP5 confocal microscope (20 spheroids per hydrogel).

Bray L.J., Secker C., Murekatete B., Sievers J., Binner M., Welzel P.B, & Werner C. (2018). Three-Dimensional In Vitro Hydro- and Cryogel-Based Cell-Culture Models for the Study of Breast-Cancer Metastasis to Bone. Cancers, 10(9), 292.

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Immunofluorescence Assay for DNA Damage Response

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Multitest slides (10 well, MP Biomedicals) were treated for 20’ with Poly-L-lysine solution (Sigma-Aldrich) at 1mg/ml concentration. After two washes with DPBS solution, approximately 0.5/1x10⁵ cells were seeded on covers for 20’ and fixed with 4% paraformaldehyde (Santa Cruz Biotechnology) for other 20’. Cells were then permeabilized with 0.1% Triton X-100. After blocking with 0.5% BSA and 0.2% fish gelatin in DPBS, cells were probed with the indicated primary antibodies. After primary antibodies incubation (53BP1 Antibody, Bethyl Laboratories; Anti-phospho Histone H2A.X (Ser139) Antibody, clone JBW301, Merck), cells were washed three times with DPBS and incubate with Alexa 488-, 568- and/or 647-labeled secondary antibodies (Invitrogen). Nuclear DNA was stained with DAPI at 0.2 μg/ml concentration (Sigma-Aldrich) and covers were mounted with Aqua-Poly/Mount solution (Polysciences. Inc.) on glass slides (Bio-Optica). Fluorescent images were acquired using Leica SP2 and Leica SP5 Confocal microscopes. Quantification of DDR foci in immunofluorescence images was conducted using Cell Profiler (version 2.1.1, revision 6c2d896) in Figures 1D and 4G.

Schiroli G., Conti A., Ferrari S., della Volpe L., Jacob A., Albano L., Beretta S., Calabria A., Vavassori V., Gasparini P., Salataj E., Ndiaye-Lobry D., Brombin C., Chaumeil J., Montini E., Merelli I., Genovese P., Naldini L, & Di Micco R. (2019). Precise Gene Editing Preserves Hematopoietic Stem Cell Function following Transient p53-Mediated DNA Damage Response. Cell Stem Cell, 24(4), 551-565.e8.

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Immunohistochemistry of Murine Lymph Nodes

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Mice were anesthetized with i.p. injection of ketamine and xylazine and perfused with cold 2% PFA. MandLN were harvested and fixed overnight in 4% PFA, and dehydrated in 30% sucrose overnight prior to embedding in TissueTek O.C. T. compound (Sakura) for cryostat sectioning. Slides with 10 µm-thick cryosections were mounted with ProLong TM Gold Antifade Mountant (Molecular Probes). Fluorescence microscopy was performed using Leica SP5 confocal microscope with 20X (NA 0.7) and 63X (NA 1.3) Leica objectives. Images were processed using Adobe Photoshop CS6 and Imaris 8.4.1 (Bitplane). Brightness and contrast were adjusted for each image individually.

Albuquerque J.B.d., Werdt D.v., Francisco D.S., Geest G.v., Altenburger L.M., Abe J., Ficht X., Iannacone M., Bruggmann R., Mueller C., & Stein J.V. (2020). CD8⁺ T cell priming in oral mucosa-draining lymph nodes supports systemic immunity.

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circHIPK3 Localization by FISH

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The circHIPK3 was labeled with Cy5, the DNA Oligo Probe (GenePharma, Suzhou, China) was labeled with 5-carboxyfluorescein (FAM), and the nucleus was backstained with 4,6-diamidino-2-phenylindole dihydrochloride (DAPI; GenePharma), which was used for fluorescence in situ hybridization (FISH) detection. After staining, the results were observed and the images were captured under a Leica SP5 confocal microscope (Leica, Wetzlar, Germany).

Zhang Y.J., Zhu W.K., Qi F.Y, & Che F.Y. (2023). CircHIPK3 promotes neuroinflammation through regulation of the miR-124-3p/STAT3/NLRP3 signaling pathway in Parkinson's disease. Advances in clinical and experimental medicine : official organ Wroclaw Medical University, 32(3).

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Immunofluorescence Imaging of Cells

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For immunofluorescence assays, cells were plated onto slides coated with poly-L-lysine (50 μg ml⁻¹) and incubated for 1 h at 37°C. Infection experiment were carried out at the indicated times. Cells were then fixed, blocked and stained with the indicated primary antibodies (5 μg ml⁻¹) followed by alexa488- or Rhodamine Red X-labelled secondary antibodies (5 μg ml⁻¹). Samples were examined under a Leica SP5 confocal microscope (Leica) fitted with a 63X objective. Images were acquired with sequential xyz acquisition mode scans with laser ranges of 418–473 nm for DAPI, 502–548 nm for Alexa-488, 584–644 nm for Rhodamine X and 737–779 nm for Alexa-647. Z-stacks of 2–5 μm were obtained using a maximum z-step size of 0.3 μm.

Moreno-Gonzalo O., Ramírez-Huesca M., Blas-Rus N., Cibrián D., Saiz M.L., Jorge I., Camafeita E., Vázquez J, & Sánchez-Madrid F. (2017). HDAC6 controls innate immune and autophagy responses to TLR-mediated signalling by the intracellular bacteria Listeria monocytogenes. PLoS Pathogens, 13(12), e1006799.

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Immunofluorescence Staining of Organoids

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Ten to 15 organoids were collected and fixed with 4% PFA for 20 minutes on RT. Before staining they were blocked with Blocking buffer for one hour on RT. Staining with primary antibodies was performed overnight at 4°C in staining buffer. The next day, after being washed with wash buffer (Supplementary Table S2), the secondaries were applied overnight at 4°C. After antibodies staining and washing, the organoids were stained with DAPI for 10 minutes on RT and mounted on concavity slides (Lab Scientific, Livingston, NJ, USA). After that the samples were imaged with a Leica SP5 confocal microscope (Leica, Wetzlar, Germany). A few organoids from both groups were imaged, and then representative images from each of the group were selected.

Kegeles E., Perepelkina T, & Baranov P. (2020). Semi-Automated Approach for Retinal Tissue Differentiation. Translational Vision Science & Technology, 9(10), 24.

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