Axio observer confocal microscope

Manufactured by Zeiss

The Axio Observer confocal microscope is a high-performance imaging system designed for advanced microscopy applications. It features a modular design and offers high-resolution, optical sectioning capabilities to enable detailed analysis of biological samples.

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Axio Observer.Z1 Inverted Confocal microscope ( Axio Observer.Z1 invert confocal microscope Axio Observer LSM710 confocal microscope LSM 880 Axio Observer laser scanning confocal microscope 710 confocal scanning Axio Observer.Z1 inverted microscope LSM Axio Observer Z1 confocal microscope Axio Observer Z1 confocal microscope Spinning disk Axio Observer Z1 confocal microscope LSM 780 NLO inverted Axio Observer Z1 confocal microscope LSM 880/Elyra/Axio Observer Z1 confocal microscope

14 protocols using axio observer confocal microscope

Assessing Cardiomyocyte Viability via LIVE/DEAD Assay

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The LIVE/DEAD viability/cytotoxicity assay was used to detect the % of living cells in ff_RGD-treated and control CM monolayers. A stock solution of 2 µM calcein AM and 4 µM ethidium homodimer-1 was prepared. A total of 20 µL of 2 mM ethidium homodimer-1 and 5 µL of 4 mM calcein AM were added to 10 mL of serum free medium. The stock solution was then vortexed to ensure thorough mixing. Glass-bottomed dishes with spontaneously beating hiPSC-CM cultures were treated with ff_RGD (2 mM) or serum free for 24 h control was filled with 200 µL of stock solution and incubated at 37 °C in a 5% CO₂ incubator for 20 min. In order to assess the fluorescence of the dishes, images were collected using a 10× objective on a Zeiss AxioObserver Confocal microscope equipped with a motorised stage. Analysis of the images was conducted offline using Fiji software.

Wang B.X., Kane C., Nicastro L., King O., Kit-Anan W., Downing B., Deidda G., Couch L.S., Pinali C., Mitraki A., MacLeod K.T, & Terracciano C.M. (2022). Integrins Increase Sarcoplasmic Reticulum Activity for Excitation—Contraction Coupling in Human Stem Cell-Derived Cardiomyocytes. International Journal of Molecular Sciences, 23(18), 10940.

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Visualizing Eluforsen Uptake in Primary HBE Cells

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In order to assess uptake of eluforsen by HBE cells, eluforsen was labeled with Cy5 and administered to primary HBE cultures. HBE cultures were treated from 2 to 28 days with fluorescently labeled eluforsen. After treatment, the cultures were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS; Life Technologies) and permeabilised with 0.1% Triton X-100 (Promega, Leiden, the Netherlands) in PBS. Next, the cultures were blocked with PBS + 5% bovine serum albumin (Sigma Aldrich), then incubated with Alexa Fluor 488-labeled phalloidin (1:40 in PBS + 5% bovine serum albumin, Life Technologies) to stain for F-actin, and subsequently washed four times with PBS. Hoechst nuclear staining (NucBlue Live ReadyProbes Reagent, Life Technologies) was added to visualize the nucleus. The uptake of Cy5-labeled eluforsen was assessed using an Axio Observer confocal microscope with a Zeiss laser scanning microscope exciter (Zeiss, Sliedrecht, the Netherlands). Pictures were processed using ZEN black software (Zeiss).

Beumer W., Swildens J., Leal T., Noel S., Anthonijsz H., van der Horst G., Kuiperij-Boersma H., Potman M., van Putten C., Biasutto P., Platenburg G., de Jonge H., Henig N, & Ritsema T. (2019). Evaluation of eluforsen, a novel RNA oligonucleotide for restoration of CFTR function in in vitro and murine models of p.Phe508del cystic fibrosis. PLoS ONE, 14(6), e0219182.

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Immunofluorescence Analysis of DNA Damage Response

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Cells grown on cover slips in a 24-well plate were fixed in 4% paraformaldehyde for 20 min and then treated with 0.1% Triton X-100 solution on ice for 4 min. Cells were then blocked by 3% BSA for 1 h followed by the antibody incubation at 4°C overnight. After that, cells were washed 3 × 5 min in PBS and then incubated with the fluorescently labeled secondary antibody for 1 h. DAPI was stained for 3 min for visualizing the nucleus. The slices were mounted by 90% glycerinum and images were captured by a Zeiss Axio Observer confocal microscope.
BrdU (Sigma, #B5002) incorporation assay was carried out by following the protocol provided by Cell Signaling Technology. Briefly, BrdU was diluted to a final concentration of 0.03 mg/mL with fresh DMEM and then applied onto the cells grown on slices. Cells were incubated with 1.5 M HCl followed by 5-min fixation in 70% cold ethanol. Immunostained with anti-BrdU antibody was then conducted as shown above. The antibody dilution ratio are as follows: p-ATM (#Ab81292, 1:100), p-ATM (10H11.E12) (#NB100-306, 1:1,000), NBS1 (#Ab32074, 1:100), RAD50 (#Ab89, 1:100), RPA2 (#10412-1-AP, 1:200), Goat Anti-Mouse IgG H&L (Alexa Fluor 488) (#Ab150113, 1:400), and Goat Anti-Rabbit IgG H&L (Alexa Fluor 647) (#Ab150079, 1:400).

Zhao K., Wang X., Xue X., Li L, & Hu Y. (2020). A long noncoding RNA sensitizes genotoxic treatment by attenuating ATM activation and homologous recombination repair in cancers. PLoS Biology, 18(3), e3000666.

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Immunofluorescence Imaging of VDR in pNKs

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CD56+ pNKs were isolated and stimulated overnight with CK in the presence of 1,25(OH)₂D3 as described above. Cells were surface-labelled for CD56 using FITC-anti-CD56 (clone REA196 Miltenyi Biotec), then fixed and permeabilised for VDR labelling with mouse anti-human VDR (D6 clone, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or mouse IgG2a isotype control according to the protocol described above for flow cytometry but with goat anti-mouse IgG2a-594 and goat-anti-FITC-488 (molecular probes, Fisher Scientific) secondary labelling for 30 mins in the presence of Hoechst 33342 (10 μg/ml; Fisher Scientific). After a final wash, cells were re-suspended in ProlongR Gold antifade reagent (Molecular Probes, Cell Signalling Technologies, UK) and placed under coverslip on charged microscope slides. Edges were sealed with nail varnish and 22-slice Z stack images of cells at 63x magnification, resolution 1024 × 1024 pixels per image were captured using the Zeiss LSM 880, Axio Observer confocal microscope and studied with Zen2012 software (Supplemental Figure 4).

Tamblyn J., Jeffery L., Susarla R., Lissauer D., Coort S., Garcia A.M., Knoblich K., Fletcher A., Bulmer J., Kilby M, & Hewison M. (2019). Transcriptomic analysis of vitamin D responses in uterine and peripheral NK cells. Reproduction (Cambridge, England), 158(2), 211-221.

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Fluorescent Immunohistochemistry of Ferroportin

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Tissue sections (4 μm) were cut from paraffin-blocked duodena and mounted on glass slides, dewaxed with xylene, and then rehydrated with a series of alcohols. For antigen retrieval, sections were incubated in 10 mmol/L citrate buffer pH 6.0 containing 0.05% Tween-20, heated to boiling, and maintained at 95°C for 20 minutes. Sections were then incubated in 50 mmol/L glycine/NH₄Cl for 15 minutes at room temperature to prevent endogenous fluorescence, blocked for 1 hour at room temperature with 10% fluorescence dilution buffer (1 mmol/L CaCl₂, 1 mmol/L MgCl₂, 5% goat serum, 5% fetal bovine serum, 2% bovine serum albumin, PBS), and incubated with FPN1 rabbit polyclonal primary antibody EB9 (1/500 dilution) overnight at 4°C. Subsequent to washing with PBS, the sections were incubated with secondary antibody (Molecular Probes goat anti-rabbit Alexa Fluor A594; Life Technologies, Carlsbad, CA; cat #A-11012, 1/500 dilution) for 30 minutes, washed with PBS, and incubated with DAPI counterstain for 5 minutes at room temperature. After further PBS washes, the sections were mounted in Prolong Gold antifade mountant (Life Technologies). Sections were viewed with a Zeiss LSM780 with AxioObserver confocal microscope with a Plan-Apochromat ×20/0.8 M27 objective, and images were acquired and analyzed using the Zeiss Zen software.

Fuqua B.K., Lu Y., Frazer D.M., Darshan D., Wilkins S.J., Dunn L., Loguinov A.V., Kogan S.C., Matak P., Chen H., Dunaief J.L., Vulpe C.D, & Anderson G.J. (2018). Severe Iron Metabolism Defects in Mice With Double Knockout of the Multicopper Ferroxidases Hephaestin and Ceruloplasmin. Cellular and Molecular Gastroenterology and Hepatology, 6(4), 405-427.

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Immunohistochemical Visualization of Viral Infection

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Following the experiments, histology was conducted to confirm placement of virus. Briefly, subjects were anesthetized and then transcardially perfused with 10% normal buffered formalin. Brains were taken and soaked overnight in fixative at 4º C and then incubated in a 25% sucrose solution until the brains sank. The sections (50 μm thick) were obtained on an American Optical 860 sliding microtome. Free-floating sections of rat brains were processed for immunohistochemistry. Briefly, sections were washed in PBS for 5 min followed by 3 × 10 min rinses in PBS + 0.5% triton X-100. Primary antibody diluted in PBS + 0.3% triton X-100 was applied overnight at 4ºC while shaking. Primary antibodies used were mouse anti-tyrosine hydroxylase (ImmunoStar #22941) at a 1:4000 dilution and a rabbit anti-GFP (Invitrogen #A6455, also cross reacts with EYFP) at a 1:2000 dilution. The following day, sections underwent 3 × 10 min PBS rinses and then were incubated with secondary antibodies of Alexa 555 donkey anti-mouse (Invitrogen, #A31570, 1:4000) and Alexa 488 goat anti-rabbit (Invitrogen #A11034, 1:2000) at room temperature for 2 hours while shaking. A last set of 3 × 10 min PBS rinses were applied to the sections that were then mounted onto slides and coverslipped with Prolong Gold media. Slides were visualized via Zeiss Axio Observer Confocal microscope.

Deal A.L., Bass C.E., Grinevich V.P., Delbono O., Bonin K.D., Weiner J.L, & Budygin E.A. (2020). Bidirectional control of alcohol-drinking behaviors through locus coeruleus optoactivation. Neuroscience, 443, 84-92.

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Adhesion-Induced Actin Cytoskeleton Dynamics

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Ramos cells were added to VCAM-1-coated chamber slides to allow adhesion (0.5 h) then incubated with the inhibitors (0.5 h) in serum-free medium. Cells were then stimulated (SDF1α) for the indicated time, fixed (2% PFA), permeabilized (0.5% saponin), washed and stained for F-actin using Alexa Fluor^® 488 phalloidin (33 nM). Images were taken by Zeiss AxioObserver confocal microscope under 63× magnification.

Ali A.Y., Wu X., Eissa N., Hou S., Ghia J.E., Murooka T.T., Banerji V., Johnston J.B., Lin F., Gibson S.B, & Marshall A.J. (2018). Distinct roles for phosphoinositide 3-kinases γ and δ in malignant B cell migration. Leukemia, 32(9), 1958-1969.

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Live Imaging of Embryonic Specimens

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For live imaging, embryos were anaesthetized using 0.016% tricaine (Sigma). Both live and fixed embryos were mounted in 0.6% low-melting agarose. Fluorescent image acquisition was performed using a Zeiss LSM exciter on an Axio Observer confocal microscope. Confocal stacks were processed for maximum intensity projections with Zeiss ZEN2009 software or ImageJ software. Images were adjusted for brightness and contrast using ImageJ. 3D reconstructions and movies were assembled using ImageJ.

Castillo-Robles J., Ramírez L., Spaink H.P, & Lomelí H. (2018). smarce1 mutants have a defective endocardium and an increased expression of cardiac transcription factors in zebrafish. Scientific Reports, 8, 15369.

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BrdU Incorporation Assay Protocol

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BrdU (Sigma, #B5002) incorporation assay was carried out by following the protocol. Briefly, BrdU was diluted to a final concentration of 0.03 mg/mL with fresh DMEM and then applied onto the cells grown on slices. Cells were incubated with 1.5 M HCl followed by 5-min fixation in 70% cold ethanol. Fluorescence staining with anti-BrdU antibody was then conducted. The slices images were captured by a Zeiss Axio Observer confocal microscope.

Wang Y., Guo Y., Duan C., Li J., Ji S., Yan H., Liu Y, & Zhang Y. (2022). LncGSAR Controls Ovarian Granulosa Cell Steroidogenesis via Sponging MiR-125b to Activate SCAP/SREBP Pathway. International Journal of Molecular Sciences, 23(20), 12132.

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Immunofluorescence Staining of Macrophages

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After co-culture, T cells were gently washed off and BMMs were fixed with 2.5% Paraformaldehyde. Slides were washed, blocked with Fc blocker (Innovex), 4% mouse serum (ImmunoReagents) and 4% goat serum. The primary antibody used was rat anti-F4/80 (Abcam cat#ab6640) at 1:500 dilution. Secondary antibodies used were AF568-conjugated goat anti-rat (Abcam cat#ab175476) at 1:1000 dilution, and AF488 conjugated chicken anti-GFP (Abcam cat#ab13970) at 1:5000 dilution. Slides were stained with Hoechst 33342 (Molecular Probes) for 30 min at 1:2000 dilution and mounted with ProLong Gold (Invitrogen). Images were acquired using the Zeiss AxioObserver confocal microscope and analyzed using ImageJ.

Zayats R., Mou Z., Yazdanpanah A., Gupta G., Lopez P., Nayar D., Koh W.H., Uzonna J.E, & Murooka T.T. (2023). Antigen recognition reinforces regulatory T cell mediated Leishmania major persistence. Nature Communications, 14, 8449.

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