Sp5 confocal system

Manufactured by Leica camera

Sourced in France

The Leica SP5 confocal system is a high-performance microscope designed for advanced imaging applications. It features a multi-channel detection system, enabling simultaneous acquisition of multiple fluorescent signals. The system provides high-resolution, 3D imaging capabilities, allowing users to visualize and analyze complex biological samples with precision.

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Lab products found in correlation

Hoechst 33342, by Thermo Fisher Scientific (4 mentions) Epifluorescence microscope, by Leica camera (2 mentions) Dmi600b fluorescence microscope, by Leica camera (2 mentions) Fluorescent secondary antibody, by Jackson ImmunoResearch (2 mentions) Alexa conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions) Alexa fluor 488 goat anti mouse, by Thermo Fisher Scientific (2 mentions) Anti lamin a c, by Santa Cruz Biotechnology (2 mentions) Anti nanog, by Abcam (2 mentions) Anti ki67, by ZSGB-BIO (2 mentions) Donkey serum, by Jackson ImmunoResearch (2 mentions) Coverslip, by Thermo Fisher Scientific (2 mentions) Anti hp1α, by Cell Signaling Technology (2 mentions) Anti sox2, by Santa Cruz Biotechnology (2 mentions) Anti h3k9me3, by Abcam (2 mentions) Mitotracker green, by Thermo Fisher Scientific (1 mentions) Ab21176, by Abcam (1 mentions) Ab150076, by Abcam (1 mentions) Anti β galactosidase 40 1a, by Developmental Studies Hybridoma Bank (1 mentions) Anti phospho histone h3, by Merck Group (1 mentions) Digital microscope, by Leica camera (1 mentions)

51 protocols using sp5 confocal system

Confocal Microscopy of Au@DTDTPA-Cy5 in U87 MG Cells

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Confocal microscopy experiments were performed with a LEICA SP5 confocal system located at the Centre de Photonique Bio-Medical (CPBM), University Paris-Sud (Orsay, France) in a controlled chamber. The samples were kept at 37 °C and regulated in CO₂. The U87 MG cells were incubated with 0.5 mM of Au@DTDTPA-Cy5 for 6 h. Cyanine 5 was excited at 633 nm and the fluorescence emission was detected in the 650–750 nm range. The images were processed with the freely available ImageJ software.
For colocalization studies, the U87 MG cells were incubated for 12 h with 200 nM MitoTracker Green (Invitrogen – Thermofisher Scientific, Waltham, MA USA). The trackers were washed out with PBS 1× before incubation for 6 h with 1 mM of Au@DTDTPA-Cy5. Organic dye was excited at 633 nm and the fluorescence emission was detected in the 650–750 nm range. The MitoTracker Green was excited at 488 nm and the fluorescence emission was detected in the 505–600 nm range. Images were recorded at three different depths (z-axis positions).

Jiménez Sánchez G., Maury P., Stefancikova L., Campion O., Laurent G., Chateau A., Bouraleh Hoch F., Boschetti F., Denat F., Pinel S., Devy J., Porcel E., Lacombe S., Bazzi R, & Roux S. (2019). Fluorescent Radiosensitizing Gold Nanoparticles. International Journal of Molecular Sciences, 20(18), 4618.

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Fluorescence Microscopy Imaging Protocol

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After immunohistochemistry or in situ hybridization experiments, images were obtained using a Nikon epifluorescence microscope, a Leica DMI600B fluorescence microscope or a Leica SP5 confocal system.

Esteves de Lima J., Bonnin M.A., Birchmeier C, & Duprez D. (2016). Muscle contraction is required to maintain the pool of muscle progenitors via YAP and NOTCH during fetal myogenesis. eLife, 5, e15593.

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Immunofluorescence Staining of Tissue Sections

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Immunostaining of tissue sections was done using a primary Rabbit Anti-Firefly Luciferase antibody (Abcam product # ab21176) and a Donkey Anti-Rabbit IgG secondary conjugated to Alexa Fluor 594 (Abcam product # ab150076). 15-micron sections of the tissue were placed on slides and kept at −80 °C. The slides were then washed using TBS plus 0.05% Triton X-100 buffer. Samples were blocked in 10% goat serum with 1% BSA. Primary antibody (12 hours at 4 °C) was used per manufacture’s recommended dilution. After washing, the secondary antibody was added at the manufacture’s recommended concentration for 1 hour at room temperature. Fluorescent imaging was done using a Leica SP5 confocal system.

Bishawi M., Roan J.N., Milano C.A., Daneshmand M.A., Schroder J.N., Chiang Y., Lee F.H., Brown Z.D., Nevo A., Watson M.J., Rowell T., Paul S., Lezberg P., Walczak R, & Bowles D.E. (2019). A normothermic ex vivo organ perfusion delivery method for cardiac transplantation gene therapy. Scientific Reports, 9, 8029.

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Immunostaining of Drosophila Intestines

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Intact fly intestines were dissected and fixed as described previously (Meng and Biteau, 2015 (link)). The Sox100B antibody was kindly provided by Steve Russell (1:1,000 dilution), and Pdm1 antibody was kindly provided by Xiaohang Yang and Cai Yu (1:1,000 dilution). Sox21a antibody was generated in the lab (1:5,000 dilution). The anti-Delta (C594.9B; 1:100 dilution), anti-Armadillo (N2 7A1; 1:100 dilution), anti-Prospero (MR1A; 1:250 dilution), anti-β-galactosidase (40-1a; 1:100 dilution) were obtained from the Developmental Studies Hybridoma Bank and the Anti-phospho-Histone H3 (06-570; 1:2,000 dilution) from Millipore. Fluorescent secondary antibodies were obtained from Jackson Immunoresearch. Hoechst 33258 was used to stain DNA.
Confocal images were collected using a Leica SP5 confocal system and processed using the Leica software, Fiji, and Adobe Photoshop CC.
For all quantifications of cell numbers, cell proportion or signal intensity, the data are represented as average ± SEM and p values are calculated using an unpaired two-tailed Student's t test unless stated otherwise.

Meng F.W., Rojas Villa S.E, & Biteau B. (2020). Sox100B Regulates Progenitor-Specific Gene Expression and Cell Differentiation in the Adult Drosophila Intestine. Stem Cell Reports, 14(2), 226-240.

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Quantifying Neuromuscular Synapse Morphometry

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Animals were fixed and subjected to antibody labeling as described^{4 (link)}. Fixed animals were treated with anti-myotactin antibodies (MH46, Developmental Studies Hybridoma Bank) at 1:50 dilution in B-PBST (PBS + 0.1% Triton-X100 + 3% BSA), incubated overnight at 4°C, and washed several times in B-PBST. Next, samples were treated with an anti-mouse secondary antibody (Alexa567-tagged, 1:2000, LifeTechnologies) overnight, washed in B-PBST, and mounted in VectorShield, cured for several hours (4°C), and imaged in a Leica SP5 confocal system. Interpunctum interval (IPI) was determined from intensity profiles, as described^{60 (link)}. The binsize for IPI histograms was calculated according to Freedman-Diaconis rule: width=3.49N^0.3, where N is the number of observations. Histograms were normalized to yield a probability density function, which were fit by a Gamma-distribution.

Krieg M., Dunn A.R, & Goodman M.B. (2014). Mechanical Control of the Sense of Touch by β Spectrin. Nature cell biology, 16(3), 224-233.

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Immunofluorescent Analysis of OGT and RL2 in Mouse Tissues

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Mice were fasted for 6 hours before sacrifice. Tissue samples were dissected, fixed in 4% paraformaldehyde for 48 hours, and embedded in paraffin blocks. H&E stains were performed by the Histology Core in the Department of Comparative Medicine. For immunofluorescent staining, mice were anesthetized for intracardial perfusion of PBS, followed by 4% paraformaldehyde with 0.08% glutaraldehyde. Brain and adipose depots were dissected and postfixed in 4% paraformaldehyde overnight. Coronal brain sections (50 μm) were prepared using a vibratome. Tissue sections were blocked with 3% BSA and 0.2% Triton X-100 in 0.1 M phosphate-buffered saline (PBS) and were incubated with primary antibodies (1:100 dilution; CST, anti-OGT, 24083; Abcam, anti-RL2, ab2739) overnight and secondary antibodies [1:400; Alexa Fluor 488 anti-rabbit immunoglobulin G (IgG), Alexa Fluor 594 anti-rabbit IgG, Alexa Fluor 594 anti-mouse IgG, and Alexa Fluor 647 anti-rabbit IgG] for 1 hour. Immunofluorescent staining and clearance of tissues were performed following previous literature (72 ). The slides were mounted with VECTASHIELD antifade mounting medium (Vector Laboratories) and saved at 4°C until imaging. A Keyence digital microscope and the Leica SP5 confocal system were used for H&E stain imaging and fluorescent imaging.

Wang Q., Zhang B., Stutz B., Liu Z.W., Horvath T.L, & Yang X. (2022). Ventromedial hypothalamic OGT drives adipose tissue lipolysis and curbs obesity. Science Advances, 8(35), eabn8092.

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Quantitative Imaging of Neuronal Dendrites

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Larvae immunostaining was described previously (Ye et al., 2011 (link)). Confocal imaging was done with a Leica SP5 confocal system with 20x or 63x glycerol immersion lenses. To minimize the variation, we only imaged neuronal dendrites and presynaptic terminals of C3 da or C4 da neurons in abdominal segments 4, 5, and 6. Images were collected with z stacks of 1-μm-step size for dendrites and 0.3-μm-step size for axons. The resulting three-dimensional images were projected into two-dimensional images by maximum projection. The same imaging settings were applied throughout the imaging process.
The Neurolucida software was used to quantify axon terminals of single C4 da neurons. Branches shorter than 5 μm were excluded from the analysis. For quantifying the number of longitudinal axonal branches visible between abdominal segment 4–6 (i.e., the connectives) of C4 da neurons, only complete connectives spanning neighboring segments were quantified. Fasciculated connectives were counted as two.
To eliminate experimenter’s bias, these experiments were carried out in double-blind fashion. The images acquired by the primary experimenter were coded and randomized by another lab member. After the primary experimenter quantified the data, the data were decoded for statistical analysis.

Liu H., Pizzano S., Li R., Zhao W., Veling M.W., Hu Y., Yang L, & Ye B. (2020). isoTarget: A Genetic Method for Analyzing the Functional Diversity of Splicing Isoforms In Vivo. Cell reports, 33(6), 108361.

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Immunostaining and Imaging of Drosophila Larvae

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Immunostaining of third-instar larvae was accomplished as previously described (Ye et al., 2011 (link)). Antibodies used include chicken anti-GFP (Aves, Tigard, Oregon) and rabbit anti-RFP (Rockland, Limerick, Pennsylvania). Samples were dehydrated and mounted with DPX mounting media (Electron Microscopy Sciences, Hatfield, Pennsylvania). Confocal imaging was completed with a Leica SP5 confocal system equipped with a resonant scanner and 63× oil-immersion lens (NA = 1.40). Images were collected and quantified as previously described (Kim et al., 2013 (link)).

Sterne G.R., Kim J.H, & Ye B. (2015). Dysregulated Dscam levels act through Abelson tyrosine kinase to enlarge presynaptic arbors. eLife, 4, e05196.

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Microscopic Imaging of Tissue Samples

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For bright-field microscopy, histological sections were imaged using a slide scanner (Nanozoomer 2.0-HT C9600, Hamamatsu) 20× objective or captured with a CoolSnap camera mounted on a bright-field microscope (Provis, Olympus). For illustration purposes, images from the nanozoomer were exported in Tiff format using the NDP View2 software (Hamamatsu).
For fluorescence microscopy, images were acquired with a Leica DM 6000B system using a 40×/0.70 oil objective (tissue cultures) or a Leica SP5 confocal system (colocalization and fiber density analyses) equipped with an Argon laser (for the 488-nm excitation), a diode 561 nm and HeNe 633 nm. Z-stacks of confocal images were acquired at 1024 × 1024-pixel resolution, with a pinhole set to one Airy unit and optimal settings for gain and offset.

Teng T., Gaillard A., Muzerelle A, & Gaspar P. (2017). EphrinA5 Signaling Is Required for the Distinctive Targeting of Raphe Serotonin Neurons in the Forebrain. eNeuro, 4(1), ENEURO.0327-16.2017.

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Immunofluorescent Localization of SIRT1

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Cells were fixed in situ with 4% PFA, washed with PBS, and permeabilized with 0.1% Triton X-100. After blocking, cells were incubated with primary antibody against SIRT1 (Abcam #32424) overnight at 4 C. The following day, cells were washed, incubated with FITC-labeled secondary antibody for 2 h, re-washed, and nuclei were labeled with DAPI. Samples were mounted and analyzed on a Leica SP5 confocal system.

Duncan M.T., DeLuca T.A., Kuo H.Y., Yi M., Mrksich M, & Miller W.M. (2016). SIRT1 is a critical regulator of K562 cell growth, survival, and differentiation. Experimental cell research, 344(1), 40-52.

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