Sp8 instrument

Manufactured by Leica

The SP8 instrument is a confocal microscope designed for high-resolution imaging and analysis of biological samples. It features advanced optics, a versatile configuration, and state-of-the-art detection capabilities to enable researchers to capture detailed visual data.

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Lab products found in correlation

Prolong gold antifade reagent, by Thermo Fisher Scientific (3 mentions) Maxblock blocking medium, by Active Motif (2 mentions) 8 well glass slides, by Merck Group (1 mentions) 4 well glass slides, by Merck Group (1 mentions) Las x software, by Leica (1 mentions)

Close spelling variants of this product

TCS SP8 instrument (3 citations)

4 protocols using sp8 instrument

Visualizing PBL34-BRX Interaction in Planta

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To detect the PBL34-BRX interaction in planta, 35S::PBL34-cYFP and 35S::nYFP-BRX were transiently co-expressed in Nicotiana benthamiana leaves, and the eYFP signal was detected by confocal microscopy using a Leica SP8 instrument with excitation 506 nm and emission 530 nm. 35S::PBL39-cYFP was used as a negative control. 35S::BRX-CITRINE, 35S::PBL34-CITRINE and 35S::PBL39-CITRINE were assayed to monitor the localization of the individual proteins.

Wang Q., Aliaga Fandino A.C., Graeff M., DeFalco T.A., Zipfel C, & Hardtke C.S. (2023). A phosphoinositide hub connects CLE peptide signaling and polar auxin efflux regulation. Nature Communications, 14, 423.

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Immunofluorescence Staining of MEFs

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MEFs were plated on 8-well glass slides (EMD Millipore), and allowed to adhere for at least 24 hr before use in experiments. Following IFN treatment, cells were fixed with freshly-prepared 4% (w/v) paraformaldehyde, permeabilized in 0.2% (v/v) Triton X-100, blocked with MAXblock™ Blocking Medium (Active Motif), and incubated overnight with primary antibodies at 4°C. After three washes in PBS, slides were incubated with fluorophore-conjugated secondary antibodies for 1 hr at room temperature. Following an additional three washes in PBS, slides were mounted in ProLong Gold antifade reagent (Invitrogen) and imaged by confocal microscopy on a Leica SP8 instrument. Primary antibodies were used at the following dilutions: anti-phospho mMLKL (1:7000), anti-cleaved caspase-8 (1:500).

Ingram J.P., Thapa R.J., Fisher A., Tummers B., Zhang T., Yin C., Rodriguez D., Guo H., Lane R., Williams R., Slifker M.J., Basagoudanavar S.H., Rall G.F., Dillon C.P., Green D.R., Kaiser W.J, & Balachandran S. (2019). ZBP1/DAI Drives RIPK3-Mediated Cell Death Induced by Interferons in the Absence of RIPK1. Journal of immunology (Baltimore, Md. : 1950), 203(5), 1348-1355.

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Visualizing LAMP1-GFP in Salmonella-infected Cells

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An expression vector encoding LAMP1-GFP fusion protein (Addgene) was retrovirally transduced into immortalized wild-type MEFs, and populations stably expressing LAMP1-GFP were obtained by selection in hygromycin. These cells were plated on 4-well glass slides (Millipore). After infection with Salmonella and/or treatment with IFN-γ, the cells were fixed with 4% (w/v) paraformaldehyde, permeabilized in 0.2% (v/v) Triton-X, and blocked with 3% (w/v) BSA in PBS containing 0.1% Triton-X. Cells were then incubated overnight at 4°C with anti-GFP and/or anti-GBP2 antibodies. After 3× washes in PBS, samples were incubated with fluorophore conjugated secondary antibodies for 1 h at room temperature. Following an additional 3× washes in PBS, cells were mounted in Pro-long Gold antifade reagent (Invitrogen) and imaged by confocal microscopy on a Leica SP8 instrument.

Ingram J.P., Tursi S., Zhang T., Guo W., Yin C., Wynosky-Dolfi M., van der Heijden J., Cai K.Q., Yamamoto M., Finlay B.B., Brodsky I.E., Grivennikov S.I., Tükel C, & Balachandran S. (2018). A Non-Pyroptotic Interferon-γ-Triggered Cell Death Mechanism in Non-Phagocytic Cells Promotes Salmonella Clearance in vivo. Journal of immunology (Baltimore, Md. : 1950), 200(10), 3626-3634.

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Immunofluorescence Quantification of Z-RNA and A-RNA

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Cells were plated on eight-well glass slides (EMD Millipore) and allowed to adhere for at least 24 h before use in experiments. As previously described (45 (link)), cells were fixed for 10 min with freshly prepared 4% (wt/vol) paraformaldehyde in PBS, permeabilized in 0.5% (vol/vol) Triton X-100 in PBS, blocked with MAXblock Blocking Medium (Active Motif), and incubated overnight with primary antibodies at 4 °C. After three washes in PBS, slides were incubated with fluorophore-conjugated secondary antibodies for 1 h at room temperature. Following an additional three washes in PBS, slides were mounted in ProLong Gold antifade reagent (Thermo Fisher Scientific) and imaged by confocal microscopy on a Leica SP8 instrument. Thirty cells from duplicated wells were selected randomly and detected the fluorescence intensity of Z-RNA (red channel) and A-RNA (green channel). Mean fluorescence intensity was quantified using Leica LAS X software. Primary antibodies were used for immunofluorescence studies: Z-RNA (clone Z22, Absolute Antibody; dilution: 1:200), A-RNA (clone 9D5; Millipore; dilution: 1:50).

Rodriguez D.A., Quarato G., Liedmann S., Tummers B., Zhang T., Guy C., Crawford J.C., Palacios G., Pelletier S., Kalkavan H., Shaw J.J., Fitzgerald P., Chen M.J., Balachandran S, & Green D.R. (2022). Caspase-8 and FADD prevent spontaneous ZBP1 expression and necroptosis. Proceedings of the National Academy of Sciences of the United States of America, 119(41), e2207240119.

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