Sp8 fluorescent microscope

Manufactured by Leica

The Leica SP8 is a confocal fluorescent microscope. It allows for high-resolution imaging of fluorescently labeled specimens. The SP8 is capable of multi-channel detection and can perform advanced techniques such as FRAP and FLIM.

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Lab products found in correlation

5 ethynyl 2 deoxyuridine (edu), by RiboBio (1 mentions) Vybrant phagocytosis assay kit, by Thermo Fisher Scientific (1 mentions) 96 well plate, by Corning (1 mentions) Fish assay kit, by Abnova (1 mentions)

Close spelling variants of this product

Fluorescent microscope (451 citations) SP8 microscope (288 citations) TCS SP8 fluorescent confocal microscope (6 citations)

5 protocols using sp8 fluorescent microscope

Atherosclerosis Analysis in Acacetin-Treated Mice

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Mice were subcutaneously injected with acacetin or normal saline two times every day for 12 weeks. Mice were made to fast after the last injection and then sacrificed. The aortic roots were embedded in OCT (Sakura, USA) and quickly frozen horizontally to −20℃. Eight micrometers serial sections of the aortic root were collected on 10 slides.
For atherosclerosis analysis, the entirety of the aorta was fixed in 4% paraformaldehyde, opened longitudinally, and then analysed en face. The aortic root slides were determined by Oil Red O (ORO) staining and quantification by Image J software as described previously.¹⁹For plaque component analysis, immunohistochemistry was carried out using Abcam's IHC staining protocol for frozen sections. Briefly, the aortic root slides were fixed in precooled acetone and then stained with anti‐iNOS, anti‐CD206, anti‐pNrf2^S40, anti‐Nrf2 and anti‐MsrA primary antibodies followed by donkey anti‐rabbit (Alexa Fluor® 488) or anti‐mouse (Alexa Fluor® 647) secondary antibodies, respectively. Images were captured using the Leica SP8 fluorescent microscope.

Wu Y., Song F., Li Y., Li J., Cui Y., Hong Y., Han W., Wu W., Lakhani I., Li G, & Wang Y. (2020). Acacetin exerts antioxidant potential against atherosclerosis through Nrf2 pathway in apoE−/− Mice. Journal of Cellular and Molecular Medicine, 25(1), 521-534.

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Visualization of DNA Replication in PSCs

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EdU, a thymidine nucleoside analog, can be used to detect cellular DNA replication activity during DNA replication in living cells through its specific reaction with Apollo^® fluorescent dyes. When the PSCs grew to approximately 80% density in 10-cm culture plates, they were transferred to 12-well plates. TCONS_00323213 knockdown and overexpression were performed when the cell density reached 40–50%. The EdU (RiboBio, Guangzhou, China) reagent was added to each well at a final concentration of 50 μM for 1.5–2 h. Then, the cells were fixed with 4% paraformaldehyde solution and 0.5% Triton X-100 and subsequently incubated with apollo staining solution for 30 min at 25 °C and protected from light. Nuclei were lightproof-stained with 4′,6-diamidino-2-phenylindole (DAPI) for 10 min and finally examined and photographed under a Leica SP8 fluorescent microscope.

Li M., Liu Q., Xie S., Fu C., Li J., Tian C., Li X, & Li C. (2023). LncRNA TCONS_00323213 Promotes Myogenic Differentiation by Interacting with PKNOX2 to Upregulate MyoG in Porcine Satellite Cells. International Journal of Molecular Sciences, 24(7), 6773.

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Quantitative Phagocytosis Assay for Macrophages

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In vitro phagocytosis assay was performed using Vybrant Phagocytosis Assay Kit (V6694, Thermo) according to the manufacturer’s protocol. Briefly, macrophages were seeded with 5000 cells per well in a 96 well plate (Corning, Tewksbury, MA, USA) in complete RPMI-1640 medium overnight, and then medium was replaced by 100 µl of fluorescent bioparticle suspension containing fluorescent E. coli (K-12 strain) bioparticles. Cells were subsequently incubated at 37 °C for 2 h, washed twice with 1 × PBS to remove non-phagocytosed particles, resuspended in 1 × PBS and confocal imagines were captured with a Leica SP8 fluorescent microscope. For each well, at least 100 cells were imaged. Images were than analyzed using Fiji software (ImageJ) to identify single cells and quantify the percentage (%) of phagocytosis-active cells.

Jiao S., Li C., Guo F., Zhang J., Zhang H., Cao Z., Wang W., Bu W., Lin M., Lü J, & Zhou Z. (2023). SUN1/2 controls macrophage polarization via modulating nuclear size and stiffness. Nature Communications, 14, 6416.

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Fluorescence in-situ Hybridization Protocol

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FISH was performed according to the protocol of FISH assay kit (Abnova, Taiwan). Briefly, cells were placed on the slides, which were sequentially treated with methanol:acetic acid, 2× standard saline citrate, hot citric acid, and pepsin. Centromere-specific α-satellite (CEN) probes for chromosomes 1 (green) and chromosome 5 (red) were added to each slide, the DNA and probe were co-denatured and hybridized, and the slides were washed and counterstained. Cells were then viewed with a Leica SP8 fluorescent microscope.

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LUPIN-Mediated Ibrutinib Delivery Assay

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5 × 10³ SKBR3 cells were seeded in glass bottom dishes and grown for 24 h in a humidified incubator at 37 °C, 5% CO₂. Cells were then washed twice with DPBS containing magnesium and calcium. To the control cells, PNA-Py-Ibrutinib (7, 10 μM) in HBSS (0.1% BSA; with magnesium and calcium), incubated for 2 h at room temperature, was added to the cells and incubated for 30 min at 37 °C and 5% CO₂. The same was repeated for the LUPIN release, where PNA-Py-Ibrutinib (7, 10 μM), SNAP-(Ru-PNA-Mtx)NLuc-cpDHFR, 10 nM), sodium ascorbate (10 mM), and furimazine (100 μM) in HBSS (0.1% BSA; with magnesium and calcium), incubated for 2 h at room temperature, was added to the cells and incubated for 30 min at 37 °C and 5% CO₂. After 30 min, Ibrutinib-Cy3 (9, 50 nM) was added and the cells were further incubated for 30 min at 37 °C and 5% CO₂ after which the cells were washed twice with DPBS (with magnesium and calcium) and once with DMEM(-)(no phenol red). The cells were then imaged using a Leica SP8 fluorescent microscope with filter settings for Cy3 in the Leica software. Images were analyzed by image J.

Lindberg E., Angerani S., Anzola M, & Winssinger N. (2018). Luciferase-induced photoreductive uncaging of small-molecule effectors. Nature Communications, 9, 3539.

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