Sp5 2 scanning confocal microscope

Manufactured by Leica

Sourced in United States

The Leica SP5 II is a scanning confocal microscope designed for high-resolution imaging. It features a laser scanning system and optimized optics to provide detailed visualization of samples. The core function of the Leica SP5 II is to enable researchers to capture precise, high-quality images of their specimens.

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Lab products found in correlation

Orca c4742 80 12ag camera, by Hamamatsu Photonics (2 mentions) Dmi6000b microscope, by Hamamatsu Photonics (2 mentions) Hank s balanced salt solution, by Thermo Fisher Scientific (1 mentions) Fluorodish, by Thermo Fisher Scientific (1 mentions) Lysoview 633, by Biotium (1 mentions) Volocity, by Quorum Technologies (1 mentions) Igepal, by Merck Group (1 mentions) Prolong diamond, by Thermo Fisher Scientific (1 mentions) Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (1 mentions) Goat anti mouse immunoglobulin g igg, by Jackson ImmunoResearch (1 mentions) Paris kit, by Thermo Fisher Scientific (1 mentions) Fish kit, by RiboBio (1 mentions) Fluorescence conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions) Anti vinculin, by Merck Group (1 mentions) Fluorescein tyramide, by PerkinElmer (1 mentions) Cy3 tyramide, by PerkinElmer (1 mentions) Anti fluorescein hrp, by PerkinElmer (1 mentions) Anti dig hrp, by PerkinElmer (1 mentions) Fluorescein labeling mix, by Roche (1 mentions) Vectashield, by Vector Laboratories (1 mentions)

Close spelling variants of this product

SP5-II AOBS confocal laser scanning microscope TCS SP5 II scanning confocal microscope Leica TCS SP5 II STED laser scanning confocal microscope SP5-II confocal laser scanning microscope TCS SP5 II confocal laser scanning microscope SP5 AOBS Upright 2 laser scanning confocal microscope Leica TCS SP5 II AOBS confocal laser scanning microscope SP5 II STED-CW Superresolution Laser Scanning Confocal microscope SP5 II confocal microscope SP5 confocal microscope

14 protocols using sp5 2 scanning confocal microscope

Confocal Imaging of Aptamer-Cell Interactions

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For confocal imaging of the aptamers bound with the cells, 1 × 10⁵ Mel28/Mel28-PLX cells and A375/A375-PLX cells were respectively seeded onto glass coverslips in 24-well dishes and cultured for 24 h. After washing twice with cold washing buffer, the cells were incubated with FITC/Cy5-labeled aptamer (250 nM) in 400 μL of binding buffer at 4°C for 1 h in the dark. Subsequently, the cells were washed twice, fixed with 4% paraformaldehyde for 10 min, and then stained with 400 μL of DAPI for 5 min. The images were captured by a Leica SP5 II scanning confocal microscope (Leica, Bannockburn, IL, USA).
For confocal imaging of the co-localization of the LL4A aptamer and the anti-CD63 antibody, Mel28-PLX cells were directly labeled with Cy5-LL4A and then incubated with anti-CD63 monoclonal antibodies at a 1:100 dilution for 2 h at 37°C. The cells were then incubated with a 1:100 dilution of TRITC (Tetramethylrhodamine-5-(and-6)-isothiocyanate)-conjugated goat anti-mouse immunoglobulin G (IgG; Jackson ImmunoResearch Laboratories) for 1 h at 37°C in the dark. The images were captured using a Leica SP5 II scanning confocal microscope (Leica, Bannockburn, IL, USA).

Li H., Liu J., Xiao X., Sun S., Zhang H., Zhang Y., Zhou W., Zhang B., Roy M., Liu H., Ye M., Wang Z., Liu-Smith F, & Liu J. (2019). A Novel Aptamer LL4A Specifically Targets Vemurafenib-Resistant Melanoma through Binding to the CD63 Protein. Molecular Therapy. Nucleic Acids, 18, 727-738.

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Immunofluorescence and 3D Imaging of Nucleoli

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Cells were washed with PBS, fixed in 4% PFA for 15 minutes and permeabilized with 0.5% PBS/Triton for 5 minutes. Incubation with primary antibody was performed for 1 h in PBS-5% BSA and cells were stained with AlexaFluor 488/568 conjugated anti-rabbit or -mouse secondary antibodies (Molecular Probes) and counterstained with DAPI. After mounting in 50% glycerol/50% 0.2 M Na-glycine, 0.3 M NaCl, 3D epifluorescent 3D image stacks were acquired using a Leica DMI6000B microscope equipped with an Orca C4742-80-12AG camera (Hamamatsu) and Volocity (Perkin-Elmer Improvision) and were subsequently deconvoluted (Iterative Restoration, Volocity). In a few cases 3D stacks were also obtained using a Leica SP5 II scanning confocal microscope. Nucleolar statistics were obtained from three independent immunofluorescence experiments in which ∼20 nuclei were analysed by a protocol established using the Volocity software. DAPI staining was used to define the nuclear volume and Fibrillarin staining to define the nucleoli and their individual volumes. 3D Immuno-FISH was performed as previously described [77] (link) using a Cy3 labeled fragment from the mouse rDNA, (positions 20138 to 23651 in Genbank Accession BK000964.3). Colocalization of FISH and protein signals were estimated using Volocity and given by the Pearson Global Correlation [78] .

Hamdane N., Stefanovsky V.Y., Tremblay M.G., Németh A., Paquet E., Lessard F., Sanij E., Hannan R, & Moss T. (2014). Conditional Inactivation of Upstream Binding Factor Reveals Its Epigenetic Functions and the Existence of a Somatic Nucleolar Precursor Body. PLoS Genetics, 10(8), e1004505.

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Cisplatin-Induced Outer Hair Cell Loss

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Postnatal days 3–5 (P3–P5) C57BL/6 mice pups were anesthetized using isoflurane and then decapitated. Using a microscope, the temporal bone was removed from the skull and cochlea was located and isolated in cold dissection media (1X Hank’s balanced salt solution (Gibco)). The lateral wall was dissected away and the whole basilar membrane containing the organ of Corti was isolated and transferred to a 35 mm tissue culture dish with a coverslip pre-coated with poly-l-ornithine and laminin. The cochlear explants were cultured in DMEM with 1% FBS and 50 µg/ml ampicillin at 37 °C in 5% CO₂ for 1 day. These explant cultures were then pre-treated with either vehicle or caffeine (100 µM) for 30 min followed by cisplatin (20 µM) for 48 hr. After the treatment end point, the cultures were fixed in 4% paraformaldehyde and stained with myosin VIIa to determine the OHC loss. One random image of 150 µm was captured from the basal turn using Leica SP5 II scanning confocal microscope, and the number of missing OHCs were counted manually. At least three independent cochlea were tested for each treatment.

Sheth S., Sheehan K., Dhukhwa A., Al Aameri R.F., Mamillapalli C., Mukherjea D., Rybak L.P, & Ramkumar V. (2019). Oral Administration of Caffeine Exacerbates Cisplatin-Induced Hearing Loss. Scientific Reports, 9, 9571.

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Quantifying Spiral Ganglion Neurons

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Decalcified cochleae were processed for cryosectioning as described previously⁵⁰. Serial mid-modiolar sections (10 μm thickness) were immunolabelled with neurofilament H antibody using standard immunofluorescence method described below to stain the SGN. The sections were then imaged using Leica SP5 II scanning confocal microscope at 40X magnification. To count the number of SGN, neurofilament H labelled cell bodies within the Rosenthal’s canal in the basal turn were counted manually from maximum intensity projection images of each section. The number of SGN counted from sections were normalized to the cross-sectional area of Rosenthal’s canal and averaged and presented as SGN count/1 mm².

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Quantifying Cochlear Hair Cell and Ribbon Synapse Damage

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The isolated rat cochleae were perfused with 4% paraformaldehyde in PBS through the oval and round window and were fixed in the same solution for 24 h at 4 °C. Cochleae were then decalcified in 120 mM EDTA (replaced daily) with constant stirring at room temperature for 3 weeks. From the decalcified cochleae the basilar membrane containing the organ of Corti was carefully microdissected and separated into three segments: apical, middle and basal turn, which corresponds to 8, 16 and 32 kHz frequencies, respectively. Microdissected cochlear whole-mount samples were labeled with antibodies against myosin VIIa, CtBP2, and GluR2 for staining of hair cells, presynaptic ribbon and post-synaptic glutamate receptors, respectively. The fluorescently labeled samples were imaged by Leica SP5 II scanning confocal microscope. In each sample, the number of missing OHCs were counted manually from ~150 OHC per turn and presented as percent loss of the total number of OHC. Ribbon synapses from the IHCs were imaged at 100X magnification to capture at least 15 IHCs per image. Paired (CtBP2 + GluR2 immunolabeling apposing each other) and orphan or unpaired (either CtBP2 or GluR2 missing) synaptic ribbons were counted manually from each image and presented as total number of synapses per IHC.

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Cellular Internalization and Lysosomal Colocalization

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Fluorescence images were taken on a Lecia SP5 II Scanning Confocal Microscope. For internalization experiments, cells were plated at a density of 50,000 cells/dish on a FluoroDish (Fisher, 35100) in 500 μL of 2% serum media. Once 60–70% confluent, cells were treated with 20nM CoGliF carried by 3:1 or 1:4 CoGliF/carrier weight ratio of PEI or GOPEI, respectively. For lysosomal staining, 1 μL of 1000x LysoView 633 (Biotium, 70058) was added to the media for 1 hr. Media was swapped after 4x washing with DPBS, and DAPI stain was added 10 minutes before imaging. Percent colocalization with lysosomes was determined using ImageJ software analysis. Background signals were excluded from the threshold in both images. A selection was created using the signal in the CoGliF channel and was superimposed onto the LysoView channel. The percent colocalization was calculated by the percentage of CoGliF pixels that overlap with LysoView 633 pixels. Data were plotted in GraphPad Prism and represent mean ± one standard deviation. T-test comparisons were used to determine the statistical significance of the resulting changes in colocalization from 0 hours post-CoGliF removal to 24 hours post removal.

Dukes M.W., Bajema E.A., Whittemore T.J., Holmgren R.A, & Meade T.J. (2022). Delivery of Targeted Co(III)-DNA Inhibitors of Gli Proteins to Disrupt Hedgehog Signaling. Bioconjugate chemistry, 33(4), 643-653.

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Immunofluorescence Imaging of Nucleolar Proteins

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Cells were cultured on poly-lysine-treated coverslips and after experimental manipulations were fixed in 4% PFA for 10 min and permeabilized with 0.25% PBS/Triton for 15 min. After a blocking step in PBS-N [PBS, 0.1% IGEPAL (Sigma)] and 5% donkey serum, cover slips were incubated with primary antibodies in PBS-N and 5% donkey serum for ∼16 h at 4°C. RPI was generally detected using a combination of anti-A194 and A135 antibodies. Cells were incubated for ∼2 h at room temperature with the appropriate AlexaFluor or Dylight 405/488/568/647 conjugated secondary antibodies (Thermo Fisher/Jackson ImmunoResearch) and counterstained with DAPI or Hoechst 33342. After mounting in Prolong Diamond (Thermo Fisher), epifluorescent 3D image stacks were acquired using a Leica DMI6000B microscope equipped with an Orca C4742-80-12AG camera (Hamamatsu) and Volocity (Quorum Technologies) or using a Leica SP5 II scanning confocal microscope as indicated. 3D image analysis and quantitation were performed with the Volocity software using DAPI or Hoechst staining to define the nuclear volume and fibrillarin staining to define the active nucleolar rDNA volume. The fluorescence background for each image channel was estimated from the average 3D signal of the image stack outside of all nuclei. The Otsu intensity threshold (41 ) was used to determine fluorescence signal significance.

Mars J.C., Tremblay M.G., Valere M., Sibai D.S., Sabourin-Felix M., Lessard F, & Moss T. (2020). The chemotherapeutic agent CX-5461 irreversibly blocks RNA polymerase I initiation and promoter release to cause nucleolar disruption, DNA damage and cell inviability. NAR Cancer, 2(4), zcaa032.

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Microscopic Analysis of Actin Cytoskeleton

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The procedure used has been described in detail in our earlier study24 (link). Briefly, A549 cells were seeded on coverslips in 6-well dishes and transfected with the miR-150 plasmid and the corresponding control vector for 48 h and then were fixed, permeabilized, blocked and stained, according to the manufacturers’ instructions, with Alexa Fluor 488 Phalloidin (Invitrogen, Carlsbad, USA). Images were captured by the Leica SP5 II scanning confocal microscope (Leica, Bannockburn, USA).

Li H., Ouyang R., Wang Z., Zhou W., Chen H., Jiang Y., Zhang Y., Li H., Liao M., Wang W., Ye M., Ding Z., Feng X., Liu J, & Zhang B. (2016). MiR-150 promotes cellular metastasis in non-small cell lung cancer by targeting FOXO4. Scientific Reports, 6, 39001.

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Subcellular Localization of BC200 by LNA-FISH

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To determine the subcellular location of BC200, locked nucleic acid-RNA fluorescence in situ hybridization (LNA-FISH) was performed with a FISH kit (RiboBio) according to the manufacturer’s protocol. LNA fluorescein-labeled probes against 18 S rRNA, U6 snoRNA, and BC200 were designed and synthesized by Ribo. Fluorescence signals were scanned using a Leica SP5 II scanning confocal microscope (Leica, Bannockburn, USA). A nucleus and cytoplasm segmentation PARIS™kit (Ambion, TX, USA) was used to segment the nucleus and cytoplasm of cells following the manufacturer’s instructions.

Liu Z., Wang P., Yuan S., Wang Y., Cao P., Wen F., Li H., Zhu L., Liang L., Wang Z., Hu B., Zheng F., Liu J., Xiao X, & Zhang J. (2022). LncRNA BC200/miR-150-5p/MYB positive feedback loop promotes the malignant proliferation of myelodysplastic syndrome. Cell Death & Disease, 13(2), 126.

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Immunofluorescence Staining Protocol

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Cells were fixed with 4% paraformaldehyde solution for 15 min, washed three times with phosphate-buffered saline (PBS) and then permeabilized cells with 0.1% Triton X-100/PBS for 5 min. The fixed preparations were blocked in 3% BSA/PBS for 1 h, incubated with primary antibodies for 1 h at room temperature, washed three times with PBS and then stained with fluorescence-conjugated secondary antibody (Invitrogen, Carlsbad, MA, USA). Specific antibodies used were summarized in the Table S2. Fluorescent images were acquired by the Leica SP5 II scanning confocal microscope (Leica, Bannockburn, USA).

Li H., Liu J., Cao W., Xiao X., Liang L., Liu-Smith F., Wang W., Liu H., Zhou P., Ouyang R., Yuan Z., Liu J., Ye M, & Zhang B. (2019). C-myc/miR-150/EPG5 axis mediated dysfunction of autophagy promotes development of non-small cell lung cancer. Theranostics, 9(18), 5134-5148.

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