Sp5x scanning confocal microscope

Manufactured by Leica camera

The Leica SP5X scanning confocal microscope is a high-performance imaging system designed for advanced research applications. It offers a combination of state-of-the-art optical capabilities, versatile configuration options, and user-friendly software. The SP5X is equipped with multiple laser lines and detectors, enabling simultaneous acquisition of multiple fluorescent signals. Its advanced scanning technology provides high-resolution, real-time imaging of live samples. The instrument is built to deliver reliable and reproducible results, making it a valuable tool for a wide range of scientific disciplines.

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5 protocols using sp5x scanning confocal microscope

Immunofluorescence Microscopy of ATRA-Treated Cells

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Cells were grown and compound-treated on glass slides in six-well cell culture plates. After 6 days of treatment with DMSO or ATRA, slides were incubated with a primary antibody at 4°C overnight (table S3), washed with phosphate-buffered saline, 0.1% tween 20 (PBST), and then incubated with a secondary antibody for 2 hours at room temperature. Secondary antibodies were conjugated with Alexa Fluor 488 (Life Technologies). Alexa Fluor 568 phalloidin (Life Technologies) and DAPI (4′,6-diamidino-2-phenylindole) (BD Biosciences) were used for counterstaining. Fluorescent images were taken with a Leica SP5X scanning confocal microscope at the Confocal and Light Microscopy core facility at the Dana-Farber Cancer Institute.

Zimmerman M.W., Durbin A.D., He S., Oppel F., Shi H., Tao T., Li Z., Berezovskaya A., Liu Y., Zhang J., Young R.A., Abraham B.J, & Look A.T. (2021). Retinoic acid rewires the adrenergic core regulatory circuitry of childhood neuroblastoma. Science Advances, 7(43), eabe0834.

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Assessing Cell Proliferation and Apoptosis in Zebrafish

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For EdU pulse labeling, Edu from the Click-iT Alexa Fluor 647 Imaging Kit (Life Technologies #C10340) was diluted to 2.5 mg/ml for retro-orbital injection. Each euthanized fish examined at 5 wpf was injected with 1 µl EdU solution, while 1.5 µl was used for 6 wpf fish. Fish were fixed 2 hours postinjection for cryo-sectioning; EdU detection was performed according to the manufacturer’s protocol.
Cryo-sectioning and immunofluorescence staining were performed as previously described.^{44 (link)} Antibodies against EGFP (Life Technologies #A6455 and #A11120), fibrillarin (Abcam #ab4566) and activated caspase-3 (BD Biosciences #559565) were used as primary antibodies. Secondary antibodies were conjugated with Alexa 488, 568, 647 (Life Technologies). DAPI (Life Technologies #S36973) was used for nuclear staining. Fluorescent images were taken by a Leica SP5X scanning confocal microscope at the Confocal and Light Microscopy core facility at Dana-Farber Cancer Institute.
Paraffin sectioning, H&E staining and immunohistochemistry with primary antibodies against TH (Pel-Freez #P40101), HuC/D (Life Technologies #A-21271) and synaptophysin (Millipore #MAB5258) were performed at the DF/HCC Research Pathology Core, and FACS at DFCI Flow Cytometry Core according to standard protocols.

Tao T., Sondalle S.B., Shi H., Zhu S., Perez-Atayde A.R., Peng J., Baserga S.J, & Look A.T. (2017). The pre-rRNA processing factor DEF is rate limiting for the pathogenesis of MYCN-driven neuroblastoma. Oncogene, 36(27), 3852-3867.

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Quantification of Apoptosis and Cell Division

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TUNEL was performed with the In-Situ Cell Death Detection kit (POD: Roche) according to the manufacturer's recommendations. Phosphorylated histone H3 labeling of fixed embryos was performed with the rabbit anti-phosphohistone H3 antibody (Santa Cruz) at 4°C overnight and visualized with Alexa-Fluor-488 goat anti-rabbit secondary antibody (Invitrogen).
Images of zebrafish immunofluorescence staining or live transgenic embryos were taken with a Leica SP5X scanning confocal microscope. The embryos were mounted in 1% low-melt agarose and the confocal images were captured with 20× objective. Fluorescence-positive cells were counted in each individual slice and sum numbers were analyzed with the GraphPad Prism 7 software using the two-tailed Student's t-test. The optical slice thickness is 3 µm.

Gjini E., Jing C.B., Nguyen A.T., Reyon D., Gans E., Kesarsing M., Peterson J., Pozdnyakova O., Rodig S.J., Mansour M.R., Joung K, & Look A.T. (2019). Disruption of asxl1 results in myeloproliferative neoplasms in zebrafish. Disease Models & Mechanisms, 12(5), dmm035790.

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EdU Pulse Labeling and Tissue Analysis

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For EdU pulse labeling, EdU from the Click-iT Alexa Fluor 647 Imaging Kit (Life Technologies #C10340) was diluted to 2.5 mg/ml for retro-orbital injection. Each killed fish examined at 5 wpf was injected with 1 μl EdU solution, whereas 1.5 μl was used for 6 wpf fish. Fish were fixed 2 h post injection for cryo-sectioning; EdU detection was performed according to the manufacturer’s protocol.
Cryo-sectioning and immunofluorescence staining were performed as previously described.^{44 (link)} Antibodies against EGFP (Life Technologies #A6455 and #A11120), Fib (Abcam #ab4566) and activated caspase-3 (BD Biosciences #559565) were used as primary antibodies. Secondary antibodies were conjugated with Alexa 488, 568, 647 (Life Technologies). DAPI (Life Technologies #S36973) was used for nuclear staining. Fluorescent images were taken by a Leica SP5X scanning confocal microscope at the Confocal and Light Microscopy core facility at Dana-Farber Cancer Institute.
Paraffin sectioning, H&E staining and immunohistochemistry with primary antibodies against TH (Pel-Freez #P40101, Rogers, AR, USA), HuC/D (Life Technologies #A-21271) and synaptophysin (Millipore #MAB5258, Billerica, MA, USA) were performed at the DF/HCC Research Pathology Core, and fluorescence-activated cell sorting at DFCI Flow Cytometry Core according to standard protocols.

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Zebrafish Immunofluorescence Staining Protocol

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Zebrafish immunofluorescence staining was performed as previously described [23 (link)]. Embryos at the indicated stage were fixed with 4% paraformaldehyde at 4°C overnight. For DNA double stand damage study in drug-treated embryos, phospho-gamma-H2A.X (Ser139) antibody (Genetex, GTX127342) and mouse anti-GFP antibody (Thermo Fisher Scientific, Catalog # MA5-15256-D680) were co-incubated at 4°C overnight and visualized with Alexa Fluor 488 goat anti-mouse secondary antibody and Alexa Fluor 546 goat anti-rabbit secondary antibody (Invitrogen). TUNEL apoptosis assay was performed on embryos with the In-Situ Cell Death Detection kit (POD: Roche) according to the manufacturer’s recommendations at 37°C for 5 hours. Zebrafish immunofluorescence staining was imaged with a Leica SP5X scanning confocal microscope with a 20x objective. Fluorescence-positive cells were counted in each individual slice, and sum numbers were analyzed with the GraphPad Prism 8 software using the two-tailed Student’s t-test. The optical slice thickness is 3 μm.

Dicker F., Quitterer U., Winstel R., Honold K, & Lohse M.J. (1999). Phosphorylation-independent inhibition of parathyroid hormone receptor signaling by G protein-coupled receptor kinases. Proceedings of the National Academy of Sciences of the United States of America, 96(10), 5476-5481.

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