Tsc sp8 confocal microscope

Manufactured by Leica

Sourced in Germany

The Leica TSC SP8 is a confocal microscope designed for high-resolution imaging of biological samples. It features a combination of advanced optics, detectors, and software to provide detailed, three-dimensional visualizations of cellular and sub-cellular structures.

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Lab products found in correlation

Las software, by Leica (4 mentions) Las x software, by Leica (3 mentions) Mowiol, by Polysciences (2 mentions) Lsm 700 confocal microscope, by Zeiss (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions) Mm af microscope, by Leica (2 mentions) Sd osr, by Olympus (2 mentions) Uapon 100xotirf 1.49 oil objective, by Olympus (2 mentions) Bovine serum albumin (bsa), by neoFroxx (1 mentions) Donkey anti rabbit 488, by Jackson ImmunoResearch (1 mentions) Dmil microscope, by Leica (1 mentions) Avertin, by Merck Group (1 mentions) Alexa fluor 488 goat anti rabbit, by Thermo Fisher Scientific (1 mentions) Sytox orange, by Thermo Fisher Scientific (1 mentions) Zen 2011, by Zeiss (1 mentions) Recombinant tgf β, by R&D Systems (1 mentions) Alexa fluor conjugated streptavidin, by Thermo Fisher Scientific (1 mentions) Prolong gold, by Thermo Fisher Scientific (1 mentions) C1006, by Beyotime (1 mentions) Quantigene viewrna ish tissue assay kit, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

SP8 confocal microscope (2556 citations) Confocal microscope (1146 citations) SP8 microscope (288 citations) TSC SP8 (76 citations) Confocal SP8 (11 citations) TSC SP8 microscope (5 citations)

35 protocols using tsc sp8 confocal microscope

Immunofluorescence Staining and Quantification

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Cellular immunofluorescence staining was performed as previously reported (Hwang et al., 2019 (link)). Cells seeded on slides were fixed with 4% PFA for 20 min at room temperature, washed with PBS, and blocked in 10% bovine serum albumin (Cat#4240GR100, BioFroxx) for 2 h in before being probed with p65 (1:500, Cat#8242, Cell Signaling Technology) or ASC (1:500, Cat#67824s, Cell Signaling Technology) antibodies. Thereafter, cells were incubated with donkey-anti-rabbit 488 (1:500, 711-545-152, Jackson) at room temperature and protected from light for 1 h. Images were acquired by Leica TSC SP8 confocal microscope. For p65 nuclear translocation analysis, Image J was used to frame nuclei and then the nuclear p65 signal fluorescence intensity was calculated. For ASC speck analysis, 5–6 slides were analyzed in each group, and ∼6–10 fields were analyzed for each slide. The percentage of ASC speck-positive cells as well as total cells per field was calculated using the Fiji software (Nagar et al., 2021 (link)).
For pyroptosis-like morphology analysis, images were acquired by Leica DMIL microscope (Leica) with the phase-contrast mode. Images were statistically analyzed using the Fiji software. Three wells (20 fields per well) of each group were detected. Percentage of pyroptosis-like blebs in total cells per field was calculated (Herr et al., 2020 ).

Xue K., Qi M., She T., Jiang Z., Zhang Y., Wang X., Wang G., Xu L., Peng B., Liu J., Song X., Yuan Y, & Li X. (2022). Argon mitigates post-stroke neuroinflammation by regulating M1/M2 polarization and inhibiting NF-κB/NLRP3 inflammasome signaling. Journal of Molecular Cell Biology, 14(12), mjac077.

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Quantifying Blood-Brain Barrier Leakage

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After 3 h of post‐MCAO reperfusion, 2% Evans Blue (0.1 mL/10 g) was injected through tail vein. One hour later, mice were anesthetized with intraperitoneal injection of 2.5% avertin (2,2,2‐tribromoethanol, Sigma‐Aldrich, 400 mg/kg body weight) and transcardially perfused with 0.9% NaCl and 4% paraformaldehyde. Brains were removed, and coronal sections of the brain were cut. After photographs of the sliced brains were captured, brain tissues were fixed in paraformaldehyde again and cryoprotected in 30% sucrose in PBS. Frozen serial coronal brain sections (25 μm thick) were prepared, mounted, and imaged by Leica TSC SP8 confocal microscope (Leica, Germany). The severity of BBB leakage was evaluated by the fluorescence intensity of Evans blue dye in the brain, as measured by the ImageJ software (version 1.43b, NIH, USA).

Xu X., Zhu L., Xue K., Liu J., Wang J., Wang G., Gu J., Zhang Y, & Li X. (2021). Ultrastructural studies of the neurovascular unit reveal enhanced endothelial transcytosis in hyperglycemia‐enhanced hemorrhagic transformation after stroke. CNS Neuroscience & Therapeutics, 27(1), 123-133.

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Immunolabeling of Brown Adipose Tissue

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Semi-thin sections of interscapular BAT were used for standard immunolabeling procedure, using a primary antibody against CAT and an appropriate fluorochrome-conjugated secondary antibody (1:400; Alexa Fluor^® 488 goat anti-rabbit, Thermo Fisher Scientific, Waltham, MA, USA). Sytox orange (1 μL ml⁻¹, Thermo Fisher Scientific, Waltham, MA, USA) was used for nuclei counterstaining. Slides were mounted with Mowiol (Polysciences, Eppelheim, Germany), and confocal images were acquired with a Leica TSC SP8 confocal microscope (Leica Microsystems, Wetzlar, Germany) using 63/1.4 NA oil immersion lens. The specificity of immunofluorescence was tested by the omission of the primary antibody.

Aleksic M., Golic I., Kalezic A., Jankovic A., Korac B, & Korac A. (2021). Hypothyroidism Intensifies Both Canonic and the De Novo Pathway of Peroxisomal Biogenesis in Rat Brown Adipocytes in a Time-Dependent Manner. Cells, 10(9), 2248.

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Immunolocalization of EGFR and EEA1

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Cells grown on 12-mm round glass coverslips were fixed, permeabilized with 0.1% Triton X-100 for 5 min at room temperature and incubated with primary and secondary antibodies as previously described [21 (link)]. Cells were viewed with a Zeiss LSM700 confocal microscope. Zen 2011 software (Carl Zeiss, Oberkochen, Germany) was used for image capture and to calculate the weighted colocalization coefficient of EGFR and EEA1.
Skin biopsies were sectioned (50 μm) using a standard cryostat (Bio-Optica, Milan, Italy). Immunohistochemistry assays were performed following a standard free-floating protocol. Fluorescent images were acquired with Leica TSC SP8 confocal microscope (Leica Microsystems, Wetzlar, Germany).

Saveri P., De Luca M., Nisi V., Pisciotta C., Romano R., Piscosquito G., Reilly M.M., Polke J.M., Cavallaro T., Fabrizi G.M., Fossa P., Cichero E., Lombardi R., Lauria G., Magri S., Taroni F., Pareyson D, & Bucci C. (2020). Charcot-Marie-Tooth Type 2B: A New Phenotype Associated with a Novel RAB7A Mutation and Inhibited EGFR Degradation. Cells, 9(4), 1028.

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Immunolabeling of Interscapular BAT

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Semi-thin sections of interscapular BAT were used for standard immunolabeling procedure, using primary antibodies against Nrf2, 4-HNE, and appropriate fluorochrome-conjugated secondary antibody (1:400; Alexa Fluor^® 488 goat anti-rabbit for Nrf2, A11008, or Alexa Fluor^® 488 goat anti-mouse for 4-HNE, A11001; Thermo Fisher Scientific, Waltham, MA, USA). DAPI (1 μL mL⁻¹, Sigma-Aldrich, Germany) was used for nuclei counterstaining. Slides were mounted with Mowiol (Polysciences, Eppelheim, Germany), and confocal images were acquired with a Leica TSC SP8 confocal microscope (Leica Microsystems) using 63/1.4 NA oil immersion lens. The specificity of immunofluorescence was tested by the omission of the primary antibody.

Aleksic M., Kalezic A., Saso L., Jankovic A., Korac B, & Korac A. (2021). The Unity of Redox and Structural Remodeling of Brown Adipose Tissue in Hypothyroidism. Antioxidants, 10(4), 591.

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Quantitative Amyloid-Microglia Colocalization

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Amyloid β and Iba-1 labelling were visualized with a Leica TSC SP8 confocal microscope (Leica Microsystems) equipped with 405/488/552 nm lasers using 40x/1.30 oil immersion lens. Images were taken with a step size of 1μm. Emission of fluorescently labelled Aβ and Iba-1 were collected sequentially. All images were taken with a 1024×1024 pixel resolution and 8-bit colour depth. Colocalization analysis was performed in ImageJ software (NIH, USA) using JACoP plugin [36 (link)] and the degree of colocalization between Iba-1 and Aβ4G8 was measured using the Pearson correlation coefficient (PCC). PCC measures the strength of a linear relationship between fluorescent intensities from the two images and produces values ranging from 1 (perfect positive correlation) to -1 (perfect inverse correlation), with 0 representing a random distribution [37 (link)].

Jović M., Lončarević-Vasiljković N., Ivković S., Dinić J., Milanović D., Zlokovic B, & Kanazir S. (2019). Short-term fish oil supplementation applied in presymptomatic stage of Alzheimer's disease enhances microglial/macrophage barrier and prevents neuritic dystrophy in parietal cortex of 5xFAD mouse model. PLoS ONE, 14(5), e0216726.

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Whole-Mount Immunostaining of Zebrafish Embryos

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For whole-mount immunostaining, deyolked embryos were fixed in 4% paraformaldehide overnight and then kept in methanol at -20 °C until staining. Embryos were rehydrated by sequential washing with gradient dilution of methanol and then permeabilized with 0.2% Triton™ X-100 in PBS (PBST) by heating at 70 °C for 15 min and blocked with 2% BSA in PBST for 2 h at room temperature. Embryos were then incubated with primary antibodies (anti-LC3, anti-p62, anti-cleaved caspase-3 or anti-cleaved PARP-1) in PBST overnight at 4 ˚C. Embryos were then washed in PBST 4 times and incubated with corresponding secondary antibodies (anti-rabbit Alexa Fluor 488, anti-rabbit Alexa Fluor 555, anti-goat Alexa Fluor 488 or anti-mouse Alexa Fluor 488) at 1:1000 dilution in PBST in dark, overnight at 4 ˚C.
Hoechst 33342 was added 2 h before the end of the incubation and samples were kept in dark at 37 ˚C with constant mixing. Stained embryos were mounted in glycerol and imaged on Leica TSC SP8 confocal microscope (Leica Microsystems) using 63/1.4 NA oil immersion lens.

Divac Rankov A., Ljujić M., Petrić M., Radojković D., Pešić M, & Dinić J. (2017). Targeting autophagy to modulate cell survival: a comparative analysis in cancer, normal and embryonic cells. Histochemistry and cell biology, 148(5).

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Monitoring Atg1 Kinase Activity in Yeast

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Atg11 deleted yeast expressing Atg13-neonGreen and either wild-type Atg1 or catalytically inactive Atg1^D211A (yAS_621 and yAS_554 respectively) were grown in YPD medium and switched to nitrogen starvation medium (SD-N). Cells were grown in SD-N medium for 3 hours before they were imaged using a Leica TSC SP8 confocal microscope. GFP was excited with an argon laser at 488 nm, and emission was recorded between 498 nm–758 nm. Cells were imaged with a 63 × /1.40 oil objective and images were acquired every 10 s for one minute after photobleaching. Images for each time point were bleach corrected and the fluorescence intensity of the bleached area was compared to the initial intensity after background subtraction.

Schreiber A., Collins B.C., Davis C., Enchev R.I., Sedra A., D’Antuono R., Aebersold R, & Peter M. (2021). Multilayered regulation of autophagy by the Atg1 kinase orchestrates spatial and temporal control of autophagosome formation. Molecular Cell, 81(24), 5066-5081.e10.

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Hippocampus and Cortex Imaging

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Images were captured used Leica TSC SP8 Confocal Microscope. DAPI was captured at an excitation peak at 359 nm and an emission peak at 457 nm, while GFAP’s fluorescence was captured at 490 nm excitation and 525 nm emission. Slides were imaged using a 20× oil objective using 4× zoom resulting at 80× magnification. Both hippocampi and the cortex were imaged for each slide. Individual nuclei and astrocytes were imaged along with colocalized regions.

Lopez K., Camacho A., Jacquez Q., Amistadi M.K., Medina S, & Zychowski K. (2022). Lung-Based, Exosome Inhibition Mediates Systemic Impacts Following Particulate Matter Exposure. Toxics, 10(8), 457.

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Myotube Differentiation Assay with Growth Factors

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C2C12 myoblasts (ATTC CRL‐1772; maintained between passages 3 and 7) were cultured in growth medium until confluency, after which were switched to differentiation medium (low glucose DMEM + 2% FBS + 1% PS) to induce differentiation to myotubes (day 0, as depicted in Fig 2A). At day 7 of differentiation, myotube cultures were separated into control (n = 5), 50 ng/ml recombinant Mstn (R&D Systems #788‐G8; n = 4), 50 ng/ml recombinant GDF11 (R&D Systems #1958‐GD; n = 5), or 50 ng/ml recombinant TGFβ (R&D Systems #240‐B; n = 3) enriched differentiation media for 72 h. All recombinant proteins were reconstituted according to the manufacturer's instruction in 4 mM HCl with 0.1% bovine serum albumin (BSA).
At day 10, cultures were fixed in 4% paraformaldehyde (PFA), permeabilized in 0.1% Triton X‐100, and blocked in 0.2% BSA for 1 h. Myotubes were stained with anti‐myosin heavy chain (clone MY‐32; Sigma‐Aldrich #M1570; St. Louis, MO) overnight, incubated with Alexa‐568‐conjugated goat anti‐mouse secondary antibody (Life Technologies #A11031; Grand Island, NY) for 1 h, and counter stained with DAPI (Sigma‐Aldrich). Images were acquired with a Leica TSC‐SP8 confocal microscope and analyzed by Leica LAS X software (200–400 myotubes per condition).

Hammers D.W., Merscham‐Banda M., Hsiao J.Y., Engst S., Hartman J.J, & Sweeney H.L. (2017). Supraphysiological levels of GDF11 induce striated muscle atrophy. EMBO Molecular Medicine, 9(4), 531-544.

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