Sp8 microscope

Manufactured by Leica Microsystems

Sourced in Germany

The Leica SP8 is a high-performance confocal microscope designed for advanced imaging applications. It features a modular design, allowing for customization to meet specific research needs. The SP8 offers a range of high-sensitivity detectors and a variety of excitation sources, enabling researchers to capture detailed, high-quality images of their samples.

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Lab products found in correlation

4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions) Leica type f immersion oil, by Leica camera (1 mentions) Leica dfc9000 gtc scmos camera, by Leica camera (1 mentions) Antifade mounting medium, by Beyotime (1 mentions) Cy3 conjugated goat anti rabbit secondary antibody, by Jackson ImmunoResearch (1 mentions) Rabbit anti p27 antibody, by Cell Signaling Technology (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Wuhan Servicebio Technology (1 mentions) Goat serum, by Beyotime (1 mentions) Ab3786, by Merck Group (1 mentions) Cc10 t 18 sc 9772, by Santa Cruz Biotechnology (1 mentions) Rabbit anti p65, by Cell Signaling Technology (1 mentions) Mouse anti gfap, by Cell Signaling Technology (1 mentions)

9 protocols using sp8 microscope

Monitoring PLK4 Reporter Translation

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A monoclonal iRFP-tagged PCNA Flp-In TRex-DLD-1 cell line was used to stably express the truncated PLK4-sfGFP-NLS reporters. Cells expressing either a uORF1+2 or a uORFs mut version of the reporter were seeded into four-chamber, 35-mm glass-bottom culture dishes (Greiner) along with 1 µg/mL doxycycline 1 d before starting the time-lapse imaging. The next day, cells were transferred to an environmental control station set to 37°C and 5% CO₂. Time-lapse movies were captured using a SP8 microscope (Leica microsystems) controlling a Leica DFC9000 GTC sCMOS camera. Images were acquired with a 40×, 1.30 NA oil objective. Every 5 min, six 3-μm z-sections were acquired in the GFP and far-red channels to observe the sfGFP-NLS and iRFP-PCNA signals, respectively. Images were acquired using Leica type F immersion oil (n = 1.5180). Approximately 3–4 h into the movie, prewarmed media containing either DMSO or TMP (to 50 µM final concentration) was carefully added to each imaging chamber.
The translation rates of uORF1+2 and uORFs mut reporters were calculated by normalizing the fluorescence intensity of nuclear-localized sfGFP in TMP-treated cells to the DMSO condition at the same time point. An additional normalization was performed to the average signal intensity from the imaging frame prior to DMSO or TMP addition.

Phan T.P., Boatwright C.A., Drown C.G., Skinner M.W., Strong M.A., Jordan P.W, & Holland A.J. (2022). Upstream open reading frames control PLK4 translation and centriole duplication in primordial germ cells. Genes & Development, 36(11-12), 718-736.

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TFEB-GFP Translocation Tracking in HEK293 Cells

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HEK293 cells transiently transfected with TFEB-GFP were seeded on coverslips. 24 h after transfection, cells were treated with 15 μM MG-132 for 0, 3, 6, and 12 h, respectively. After treatment, cells were fixed in 4% paraformaldehyde (Solarbio; P1110) for 20 min and stained with DAPI (300 nM; Thermo Fisher Scientific) at room temperature for 20 min. Cells were rinsed with PBS and sealed in antifade mounting Medium (Beyotime; P0126). Results were analyzed by using SP8 microscope (Leica Microsystems, Germany).

Li C., Wang X., Li X., Qiu K., Jiao F., Liu Y., Kong Q., Liu Y, & Wu Y. (2019). Proteasome Inhibition Activates Autophagy-Lysosome Pathway Associated With TFEB Dephosphorylation and Nuclear Translocation. Frontiers in Cell and Developmental Biology, 7, 170.

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Tracing T Cell Trafficking in Lungs

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RAG2-deficient mice were injected intravenously with 0.1 × 10⁶ KPP-eGFP cells and 4 × 10⁶ tdTomato⁺ CD3⁺ T cells isolated as described below. Then, 8 days after intravenous injection, the mice were euthanized, and 20 ml of cold PBS/heparin 5 IU ml⁻¹ solution was perfused directly into the right ventricle using a 27-gauge needle. Lungs were isolated by dissection and tissues were fixed using 4% paraformaldehyde in PBS^{23 (link)}. Tissue clearing was performed using the FluoClearBABB approach^{27 (link)} and whole-mount images were then acquired using a SP8 microscope equipped with a white-light laser and the HCX APO L ×20/0.95 NA IMM lens (Leica Microsystems). Imaging data were analysed on a workstation (Thinkmate) using Imaris software (Bitplane).

Ortiz-Muñoz G., Brown M., Carbone C.B., Pechuan-Jorge X., Rouilly V., Lindberg H., Ritter A.T., Raghupathi G., Sun Q., Nicotra T., Mantri S.R., Yang A., Doerr J., Nagarkar D., Darmanis S., Haley B., Mariathasan S., Wang Y., Gomez-Roca C., de Andrea C.E., Spigel D., Wu T., Delamarre L., Schöneberg J., Modrusan Z., Price R., Turley S.J., Mellman I, & Moussion C. (2023). In situ tumour arrays reveal early environmental control of cancer immunity. Nature, 618(7966), 827-833.

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Immunofluorescence Analysis of P27 Expression

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Human cardiac myocytes cells were seeded onto a 12‐well plate covered with slips and then transfected with WT or variant plasmid DNA. Cells were harvested 24 h after transfection. Cells were incubated with rabbit anti‐P27 antibody (dilution 1 : 800; Cell Signaling Technology) diluted in NaCl/P_i containing 5% goat serum (Beyotime) and 0.2% Triton X‐100 at 4 °C overnight, followed by incubation with Cy3‐conjugated goat anti‐rabbit secondary antibody (dilution 1 : 300; Jackson ImmunoResearch, West Grove, PA, USA). Cell nuclei were stained using 4',6‐diamidino‐2‐phenylindole (Servicebio). Image analysis [21, 22] was condicted using a SP8 microscope (Leica Microsystems, Wetzlar, Germany).

Peng J., Wang Q., Meng Z., Wang J., Zhou Y., Zhou S., Song W., Chen S., Chen A.F, & Sun K. (2021). A loss‐of‐function mutation p.T256M in NDRG4 is implicated in the pathogenesis of pulmonary atresia with ventricular septal defect (PA/VSD) and tetralogy of Fallot (TOF). FEBS Open Bio, 11(2), 375-385.

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Salmonella Colonization Patterns in Tomato

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Tomato plants were grown under sterile conditions in order to visualize the colonization patterns of Salmonella. Seven to ten-day old plants were transferred to conical tubes containing ¼-strength MS liquid medium. Plants were allowed to adapt to the medium for 24 h before the medium was inoculated with a final OD_{600 nm} = 0.1 corresponding to 10⁸ colony forming units/mL (CFU/mL) of either S. Typhimurium 14028s-GFP or S. Typhimurium ∆fljB∆fliC-GFP strain. Plants were sampled (leaves and roots) 24 h post inoculation (hpi), stained with propidium iodide (PI) solution (1 µg/ mL) for 5 min and mounted on a microscope slide in 4′, 6-diamidine-2′-phenylindole dihydrochloride (DAPI) solution (10 µg/ mL). Confocal laser scanning microscopy was performed using SP8 microscope (Leica Microsystems, Wetzlar, Germany) with excitation 405 nm, emission 430–480 nm (blue), excitation 488 nm, emission 500–550 nm (green), excitation 561 nm, emission 600–680 nm (red) including autofluorescence of chloroplasts.

Zarkani A.A., López-Pagán N., Grimm M., Sánchez-Romero M.A., Ruiz-Albert J., Beuzón C.R, & Schikora A. (2020). Salmonella Heterogeneously Expresses Flagellin during Colonization of Plants. Microorganisms, 8(6), 815.

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Subcellular Localization of C-Tail Fusion Protein

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To visualize the subcellular localization of the C-tail fusion protein, vein samples were prepared and cleared as described previously [12 (link),14 (link)]. DAPI was used to stain the nuclei before imaging. The stained samples were visualized with a SP8 microscope (Leica Microsystems CMS GmbH, Mannheim, Germany). DAPI was excited at 408 nm and detected in a window of 430–470 nm. mVENUS was excited at 514 nm and detected in a range of 520–540 nm.

Wu Q., Chen M, & Kumari A. (2023). Dual localization of the carboxy-terminal tail of GLR3.3 in sieve element–companion cell complex. Communicative & Integrative Biology, 16(1), 2167558.

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Localization of RBM5 in Adult Lung

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The localisation of RBM5 in the adult lung (8 weeks old) was determined by immunofluorescence using an RBM5 mouse monoclonal antibody as described previously^{17 (link)}. To distinguish different cell lineages in the lung, we co-labelled sections with antibodies against Pro-surfactant protein C (AB3786, Merck Millipore), as a marker for type II alveolar epithelial cells, and CC10 (CC10 (T-18), SC-9772, Santa Cruz Biotechnology), as a marker for secretory cells. Protein localisation was determined through confocal microscopy using an SP-8 microscope (Leica Microsystems).

Jamsai D., Watkins D.N., O’Connor A.E., Merriner D.J., Gursoy S., Bird A.D., Kumar B., Miller A., Cole T.J., Jenkins B.J, & O’Bryan M.K. (2017). In vivo evidence that RBM5 is a tumour suppressor in the lung. Scientific Reports, 7, 16323.

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Visualizing GLR3.3-mVENUS Subcellular Localization

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To observe the subcellular localization of GLR3.3‐mVENUS and its derivatives, vein samples were prepared from expanded leaves of 5‐wk‐old plants. Vein extraction was performed following the description in Kurenda & Farmer (2018 (link)). Isolated veins were immediately fixed with 4% paraformaldehyde (PFA) solution for 1 h with gentle shaking and then subjected to ClearSee treatment (Ursache et al., 2018 (link)) for 2 d. Refreshing the ClearSee solution was necessary to get sufficiently cleared samples. To stain samples, 0.1% (w/v) Calcofluor‐white was added into the ClearSee solution, and the samples were stained for 15 min. Then the samples were washed twice with ClearSee solution before observation. All the samples were visualized with an SP8 microscope (Leica Microsystems CMS GmbH, Mannheim, Germany). Sequential scanning mode was used to avoid interference between channels. mVENUS was excited at 514 nm and detected in the range 520–540 nm. In most cases, Chl autofluorescence still remained and was detected in an emission window of 650–700 nm. Calcofluor‐white was imaged with 405 nm excitation and 430–460 nm emission.

Wu Q., Stolz S., Kumari A, & Farmer E.E. (2022). The carboxy‐terminal tail of GLR3.3 is essential for wound‐response electrical signaling. The New Phytologist, 236(6), 2189-2201.

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Immunofluorescence Analysis of Brain Tissue and Cells

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The brain slices were laid at on an adhesive glass slide. Then the slices were permeabilized with 0.3 % Triton X-100 for 30 min, and blocked with goat serum for 1 h. For immuno uorescence of cells, after the cell slices were xed with 4 % paraformaldehyde for 15 minutes, the cell slices were treated with 0.3 % Triton X-100 for 5 min and 5 % BSA for 1 h. Brain slices and cell slices were incubated at 4°C overnight with following primary antibodies: mouse anti-GFAP (1:300; Cell Signaling Technology, Danvers, MA, USA), and rabbit anti-p65 (1:400; Cell Signaling Technology, Danvers, MA, USA). The secondary antibodies were as follows: Cy3 anti-rabbit IgG (1:100; Boster Biological Technology, Wuhan, China), FITC anti-mouse IgG (1:50; Boster Biological Technology, Wuhan, China). The immuno uorescence pictures were collected with SP8 microscope (Leica Microsystems, Germany), and the immuno uorescence density was analyzed with ImageJ software.

Xu D., Kong T., Shao Z., Zhang R., Zhang S., Kong Q., Chen J., Cheng B., & Wang C. (2021). Orexin-A Alleviates Astrocytic Apoptosis and Inflammation via Inhibiting OX1R-mediated NF-κB and MAPK Signaling Pathways in Cerebral Ischemia/reperfusion Injury.

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