710 laser scanning microscope

Manufactured by Zeiss

Sourced in Germany, United States

The Zeiss 710 laser-scanning microscope is a high-performance imaging system designed for advanced biological and materials research. It features a modular design, allowing for customization to meet specific research requirements. The microscope utilizes a laser-scanning technology to capture detailed images of samples, providing researchers with a powerful tool for analysis and visualization.

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LSM 710 Spectral Confocal Laser Scanning Microscope LSM 710 Axio Obzerver Z1 DUO NLO laser scanning microscope LSM 710 META confocal laser-scanning microscope Zeiss 710 Meta Confocal Laser Scanning Microscope LSM 710 Carl Zeiss Confocal Laser scanning microscope CLSM 710 Spectral Confocal Laser Scanning Microscope LSM 710 Confocal Laser Point-Scanning Microscope 710 laser scanning confocal microscope Inverted 710 laser scanning confocal microscope Zeiss Laser Scanning Microscope 510 Meta or 710

44 protocols using 710 laser scanning microscope

Immunofluorescent Detection of MUC5AC in SAEC ALI

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SAEC ALI cultures growing on transwell membranes were fixed in 4% paraformaldehyde overnight and embedded in paraffin. Blocks were sectioned in 3–4 µm thick slices. Paraffin-embedded tissue slides were subjected to heat-induced antigen retrieval in citrate buffer (pH 6.0, H.I.E.R, BioLegend San Diego, CA, USA) for 30 min. Mouse monoclonal Anti-MUC5AC antibody, clone 45M1, NBP2-32732AF488 (Novus Biologicals, Centennial, CO, USA) was used for immunofluorescence antigen detection. Slides were mounted in ProLong™ Diamond Antifade Mountant (Thermo Fisher Scientific, Waltham, MA, USA) and stored in the dark. Images were taken using the Zeiss Laser Scanning Microscope 710 (Carl Zeiss, Oberkochen, Germany).

Wohnhaas C.T., Gindele J.A., Kiechle T., Shen Y., Leparc G.G., Stierstorfer B., Stahl H., Gantner F., Viollet C., Schymeinsky J, & Baum P. (2021). Cigarette Smoke Specifically Affects Small Airway Epithelial Cell Populations and Triggers the Expansion of Inflammatory and Squamous Differentiation Associated Basal Cells. International Journal of Molecular Sciences, 22(14), 7646.

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Immunohistochemical Analysis of Airway Epithelium

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Cells, growing on transwell membranes (pore size of 0.4 µm), were fixed in 4% paraformaldehyde over night and embedded in paraffin. After sectioning (section slice thickness approximately 3–4 µm), slides were deparaffinized and hematoxylin and eosin (H&E) stainings were performed according to standard procedures. For immunohistochemical (IHC) investigation, paraffin-embedded tissue slides were subjected to heat induced antigen retrieval in citrate buffer (pH 6.0, H.I.E.R, BioLegend San Diego, CA, USA) for 30 minutes. Antibodies used for immunofluorescence or chromogenic antigen detection are listed in Supplementary Information, Table S2. After staining, tissues slides were mounted either with ProLong™ Diamond Antifade Mountant (Thermo Fisher Scientific, Waltham, MA, USA) or with Entellan® new (Merck KGaA, Darmstadt, Germany). For immunofluorescence samples, stained slides were stored at 4 °C in the dark and images were taken using the Zeiss Laser Scanning Microscope 710 or an AxioImager M2 (both Carl Zeiss, Oberkochen, Germany) in case of chromogenic IHC. Stainings for KRT5, MUC5AC, SCGB1A1 and acetylated Tubulin were used to assess protein expression qualitatively and representatively. Claudin-10 protein expression was semi-quantitatively analyzed using digital image analysis (compare section “Image Analysis”).

Gindele J.A., Kiechle T., Benediktus K., Birk G., Brendel M., Heinemann F., Wohnhaas C.T., LeBlanc M., Zhang H., Strulovici-Barel Y., Crystal R.G., Thomas M.J., Stierstorfer B., Quast K, & Schymeinsky J. (2020). Intermittent exposure to whole cigarette smoke alters the differentiation of primary small airway epithelial cells in the air-liquid interface culture. Scientific Reports, 10, 6257.

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NMR, MS, and Confocal Microscopy Analyses

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¹H NMR and ¹³C{¹H} spectra were collected at 25 °C on Bruker AVB-400, AVQ-400, and AV-300 spectrometers at the College of Chemistry NMR Facility at the University of California, Berkeley. All chemical shifts are reported in the standard δ notation of parts per million relative to residual solvent peak as an internal reference. Splitting patterns are indicated as follows: br, broad; s, singlet; d, doublet; t, triplet; m, multiplet; dd, doublet of doublets. Low-resolution electrospray mass spectra were recorded on a liquid chromatograph–mass spectrometer (Agilent Technology 6130, quadrupole LC/MS). High-resolution mass spectra were collected at the College of Chemistry Mass Spectrometry Facility at the University of California, Berkeley. Reaction kinetics of the probes with species of interest and other substrates were investigated by LC–MS (Agilent Technology 6130, quadrupole LC/MS) coupled with photodiode array for detection (λ = 275 nm). Confocal microscopic images were recorded on a Zeiss laser scanning microscope 710 with a 40× water-immersion objective lens using Zen 2009 software (Carl Zeiss).

Chung C.Y., Timblin G.A., Saijo K, & Chang C.J. (2018). Versatile Histochemical Approach to Detection of Hydrogen Peroxide in Cells and Tissues Based on Puromycin Staining. Journal of the American Chemical Society, 140(19), 6109-6121.

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Confocal Imaging of FIP-1 Localization

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A Zeiss laser
scanning microscope 710 with a 20x objective lens and Zen 2009 software
(Carl Zeiss) was used for all confocal fluorescence imaging experiments.
FIP-1 was excited using a 488 nm Ar laser (“Green” channel
and “FRET” channel) and 543 nm HeNe laser (red channel).
“Green” emission was collected using a META detector
between 500 and 535 nm, “FRET” emission was collected
using a META detector between 555 and 611 nm, and “red”
emission was collected using a META detector between 555 and 611 nm.
Hoechst 33342 was excited with a 405 nm diode laser, and emission
was collected using a META detector between 410 and 590 nm. Cells
were kept at 37 °C throughout imaging experiments, and HBSS (containing
calcium and magnesium) was used as the imaging buffer in all experiments.
Image analysis and quantification was performed using ImageJ (National
Institutes of Health). Quantification of fluorescence intensities
were conducted as described previously.^{66 (link)} Statistical analyses for multiple comparisons were carried out through
one-way ANOVA with the Bonferroni correction using the software R.

Aron A.T., Loehr M.O., Bogena J, & Chang C.J. (2016). An Endoperoxide Reactivity-Based FRET Probe for Ratiometric Fluorescence Imaging of Labile Iron Pools in Living Cells. Journal of the American Chemical Society, 138(43), 14338-14346.

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Immunofluorescence Analysis of Ileal Tissue

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The ileal tissues from the distal portion of the ileum were freshly isolated and paraffin-embedded after fixation with 10% neutral buffered formalin. Immunofluorescence was performed on the paraffin-embedded sections (5 μm). After preparation of the slides, as described previously (31 (link)), the tissue samples were incubated with the indicated primary antibody, anti-lysozyme (1:100, sc27958, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), at 4°C overnight. The samples were then incubated with the sheep anti-goat Alexa Fluor 594 (A11058, Life Technologies, Grand Island, NY, USA) and DAPI (D1306, Life Technologies, Grand Island, NY, USA) for 1 h at room temperature. The tissues were mounted with SlowFade (s2828, Life technologies, Grand Island, NY, USA), followed by a coverslip, and the edges were sealed to prevent drying. The specimens were examined with a Zeiss laser scanning microscope 710 (Carl Zeiss, Inc., Oberkochen, Germany).

Jiao Y., Zhang Y.G., Lin Z., Lu R., Xia Y., Meng C., Pan Z., Xu X., Jiao X, & Sun J. (2020). Salmonella Enteritidis Effector AvrA Suppresses Autophagy by Reducing Beclin-1 Protein. Frontiers in Immunology, 11, 686.

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Confocal Fluorescence Imaging of Cellular Processes

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Confocal fluorescence cell imaging was performed with a Zeiss laser scanning microscope 710 with a 40× water-immersion objective lens using Zen 2009 software (Carl Zeiss). Hoechst 33342 was excited with a 405 nm diode laser, and emission was collected on a META detector between 450 and 500 nm. Alexa Fluor 488 was excited at 488 nm with an Ar laser, and emission was collected on a META detector between 500 and 625 nm. Alexa Fluor 647 was excited with a 633 nm HeNe laser, and emission was collected on a META detector between 638 and 759 nm. PF2 was excited at 488 nm with an Ar laser, and emission was collected on a META detector between 493 and 630 nm. MitoPY1 was excited at 514 nm, and emission was collected on a META detector between 525 and 640 nm. PBS solution was used as the imaging buffer for all confocal experiments. Image analysis was performed by use of ImageJ. A region of interest (ROI) was created, and cellular fluorescence intensity was measured. The reported cellular fluorescence intensity was determined by averaging the measured intensity of five different ROIs from at least three different images in triplicate experiments. Statistical analyses were performed with a two-tailed Student’s t-test (MS Excel).

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Integrin αvβ3 Expression and Photothermal Cytotoxicity

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Human glioma cell line U87MG-luc and human breast cancer cell line MCF-7 were maintained in DMEM (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum. Integrin α_vβ₃ expression in U87MG-luc and MCF-7 cells was identified by immunohistochemical staining. The cells were incubated for 4 h with ICG, IR, or IPR (20 μM). The cytoskeleton was stained with FITC-phalloidin and nuclei were stained with DAPI. Confocal laser scanning microscopy was conducted using a Zeiss laser scanning microscope 710 (Carl Zeiss Oberkochen, Germany).
Cell Counting Kit-8 (CCK-8, Solarbio, China) was used to evaluate the cytotoxicity of ICG, IR and IPR in U87MG-luc cells after photothermal treatment. U87MG-luc cells were seeded at 1 × 10⁴ cells/well on 96-well plates and cultured for 48 h. Then, different concentrations (1, 2, 5, 10, 20, 50, and 100 μM) free PBS, ICG, IP, IR, or IPR were added to the cells, followed by incubation for 24 h. After rinsing with PBS, photothermal treatment was performed at an excitation wavelength of 808 nm for 3 min, followed by treatment with CCK-8 reagent (10 μL each well) for an additional 1 h. Absorbance was measured in a microplate reader (absorbance: 450 nm). Corresponding cells without any treatment were used as controls for three replicates.

Song W., Zhang X., Song Y., Fan K., Shao F., Long Y., Gao Y., Cai W, & Lan X. (2022). Enhancing Photothermal Therapy Efficacy by In Situ Self-Assembly in Glioma. ACS applied materials & interfaces, 15(1), 57-66.

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Ferroptosis Mitochondrial Changes with ADA-409-052

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To demonstrate the effect of ADA-409-052 on mitochondria changes upon ferroptosis, PC-12 cells were exposed to glutamate (20 mM) in presence or absence of ADA-409-052 (10 µM) for 24 h, as described before. Live cells were stained with MitoTracker Red CMXRos (ThermoFischer Scientific) and imaged with a 63X Achroplan objective on a Zeiss Laser Scanning Microscope 710 with Zen Imaging Software (Zeiss) (see suppl. information for the full protocol).

Keuters M.H., Keksa-Goldsteine V., Dhungana H., Huuskonen M.T., Pomeshchik Y., Savchenko E., Korhonen P.K., Singh Y., Wojciechowski S., Lehtonen Š., Kanninen K.M., Malm T., Sirviö J., Muona A., Koistinaho M., Goldsteins G, & Koistinaho J. (2021). An arylthiazyne derivative is a potent inhibitor of lipid peroxidation and ferroptosis providing neuroprotection in vitro and in vivo. Scientific Reports, 11, 3518.

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Immunofluorescence Staining of Cultured Cells

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Cells were plated on fibronectin-coated glass cover- slips at 100,000–300,000 cells per coverslip. Twelve-to-sixteen hours later, the slides were rinsed with PBS once and fixed for 15 min with 4% paraformaldehyde at room temperature prior to permeabilization with 0.1% Saponin for 5 min. Slides were incubated with primary antibody in 5% normal goat serum overnight at 4 degC, rinsed four times with PBS, and incubated with secondary antibodies produced in goat (diluted 1:400 in 5% normal goat serum) for 45 min at room temperature in the dark. Slides were mounted on glass slides using Vectashield (Vector Laboratories) and imaged on a Zeiss Laser Scanning Microscope (LSM) 710. Images were processed using ImageJ.

Adams C.R., Htwe H.H., Marsh T., Wang A.L., Montoya M.L., Subbaraj L., Tward A.D., Bardeesy N, & Perera R.M. (2019). Transcriptional control of subtype switching ensures adaptation and growth of pancreatic cancer. eLife, 8, e45313.

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Macrophage and MSC Responses to Titanium Surface Topography

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MSC and naïve macrophages were plated on smooth, rough, or rough-hydro Ti surfaces at a density of 10,000 cells/cm² in DMEM (ThermoFisher Scientific) with 10% fetal bovine serum (ThermoFisher Scientific), and 50 U/mL penicillin-50 μg/mL streptomycin (ThermoFisher Scientific) and incubated for 24 hours (macrophages) or 7 days (MSC). Macrophages were cultured with or without ecig-AMs with nicotine at 1:1000 dilution. After incubation, cells were washed twice with warm PBS and fixed with 4% paraformaldehyde (ThermoFisher Scientific) for 1 hour. Cell membrane was permeabilized in 0.1% Triton X-100 (Sigma–Aldrich) and cytoskeleton was then stained with phalloidin conjugated with Alexa Flour 488 and Hoechst 33342 (ThermoFisher) for nuclear staining. Cells were visualized at 3 randomly selected regions on six independent disks using a Zeiss Laser Scanning Microscope (LSM710).

Abaricia J.O., Whitehead A.J., Kandalam S., Shah A.H., Hotchkiss K.M., Morandini L, & Olivares-Navarrete R. (2021). E-cigarette Aerosol Mixtures Inhibit Biomaterial-Induced Osseointegrative Cell Phenotypes. Materialia, 20, 101241.

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