For fluorescence imaging, cells were imaged in 8-well chamber slides (ibidi) in Leibovitz L-15 media with a heated 37°C chamber or in DMEM (no phenol red) with a heated 37°C chamber in 5% CO2. Images were taken every 4 minutes with either a 20x /0.4 NA air objective using a Zeiss Axio Observer 7 with a CMOS Orca flash 4.0 camera at 4x4 binning or a 40x/1.3 NA oil objective using a DV Elite system equipped with Photometrics CascadeII:1024 EMCCD camera at 4x4 binning. For brightfield imaging, cells were imaged in a 24-well plate in DMEM in a heated chamber (37°C and 5% CO2) with a 10x/0.5 NA objective using a Hamamatsu ORCA-ER camera at 2x2 binning on a Zeiss Axiovert 200M, controlled by Micro-manager software (open source: https://micro-manager.org/ ) or with a 20x/0.4 NA air objective using a Zeiss Axio Observer 7 as detailed above. Mitotic exit was defined by cells flattening down in the presence of nocodazole and MPS1 inhibitor. In assays where both recombinant BUBR1 and KNL1 are expressed in cells, cells were selected for quantification based on high levels of Turq2 as an indication of successful transient transfection of Turq2-BUBR1 constructs into cells stably expressing YFP-tagged KNL1 constructs.
Axio observer 7
Manufactured by Zeiss
Sourced in Germany, United States
The Axio Observer 7 is a high-performance inverted microscope designed for advanced imaging applications. It features a stable, ergonomic design and offers a range of optical configurations to suit various research and analysis needs.
Automatically generated - may contain errors
Lab products found in correlation
Axiovert 200m, by Zeiss (13 mentions)
Lsm 880, by Zeiss (11 mentions)
Axiocam 506, by Zeiss (10 mentions)
Zen software, by Zeiss (10 mentions)
Zen blue software, by Zeiss (8 mentions)
Apotome 2 module, by Zeiss (6 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (6 mentions)
Apotome, by Zeiss (6 mentions)
Lsm 710 confocal microscope, by Zeiss (6 mentions)
Apotome 2, by Zeiss (5 mentions)
Axiocam712mono, by Zeiss (4 mentions)
Axiocam 702, by Zeiss (4 mentions)
Axiocam 305, by Zeiss (4 mentions)
Hoechst 33258, by Merck Group (4 mentions)
Orca er camera, by Zeiss (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions)
Zen 2.6 pro, by Zeiss (3 mentions)
Pac 1, by BD (3 mentions)
Im1759u, by Beckman Coulter (3 mentions)
Plan apochromat 63 1.4 oil dic m27 objective, by Zeiss (3 mentions)
300 protocols using axio observer 7
1
Fluorescence and Brightfield Imaging of Mitotic Exit
2
Live Cell Imaging Protocols for Yeast and HEK293 Cells
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For yeast cells, SD medium containing agar was poured into a Gene Frame (ThermoFisher Scientific) attached to a cover glass. Cells were loaded and the agar pad was sealed with a coverslip. The cells were transferred into an incubation chamber maintained at 30 °C. Images were obtained using an Axio Observer 7 fluorescence microscope (Zeiss), equipped with an Axiocam 506 mono camera and a 20×, as well as 100× oil objective lens (Plan-APOCHROMAT 100×/1.4 Oil DIC). Focus stabilization was done with the Definite Focus module (Zeiss).
For human HEK293 cells, 40,000 cells were seeded into µ-Slide 4 Well chambered coverslips (Ibidi) using medium without phenol red. Cell transfection was performed as described above using medium without phenol red. After 24 h, HEPES pH 7.4 buffer was added to a final concentration of 25 mM and the cells were transferred to a incubation chamber maintained at 37 °C. Images were obtained using an Axio Observer 7 fluorescence microscope (Zeiss), equipped with an Axiocam 506 mono camera, and a 20× objective lens.
For human HEK293 cells, 40,000 cells were seeded into µ-Slide 4 Well chambered coverslips (Ibidi) using medium without phenol red. Cell transfection was performed as described above using medium without phenol red. After 24 h, HEPES pH 7.4 buffer was added to a final concentration of 25 mM and the cells were transferred to a incubation chamber maintained at 37 °C. Images were obtained using an Axio Observer 7 fluorescence microscope (Zeiss), equipped with an Axiocam 506 mono camera, and a 20× objective lens.
3
Microscopy Techniques for Cell Migration
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Phase contrast images were acquired using a Nikon Eclipse Ts2 with x10 (ADL Ph1 0.25 NA) and x20 (ADL Ph1 0.40 NA) objectives. Migration experiments were performed by using phase contrast microscopy using an Axio Observer 7 (Carl Zeiss) using a x10 objective acquiring an image every 5 min for 6 h at 37 °C and 5% CO2. Live-cell imaging of actin and microtubules was performed using an Axio Observer 7 (Carl Zeiss) using a x63 objective. Immunostained samples were observed using confocal laser scanning microscope (LSM 800, Zeiss) equipped with a x63 oil objective (NA 1.40) and a x100 oil objective (NA 1.40).
4
Fluorescence and Brightfield Imaging of Mitotic Exit
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For fluorescence imaging, cells were imaged in 8‐well chamber slides (ibidi), on either a Zeiss Axio Observer 7 with a CMOS Orca flash 4.0 camera or a DeltaVision Elite equipped with Photometrics CascadeII:1024 EMCCD or CoolSNAP HQ (Photometrics) camera. The objectives used for fluorescent imaging were either 20×/0.8 NA or 40×/1.3NA. For brightfield imaging, cells were imaged in a 24‐well plate in DMEM on a Zeiss Axiovert 200M using Hamamatsu ORCA‐ER camera and controlled by Micro‐manager software (open source: http://micro-manager.org ), or a Zeiss Axio Observer 7 as detailed above. The air objectives used for brightfield imaging were either 10×/0.5 NA or a 20×/0.4 NA. Mitotic exit was defined by cells flattening down in the presence of nocodazole and MPS1 inhibitor.
5
Fluorescent Imaging in Biological Research
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Images in Figures 1A,B, G; 2C, G; 4D; 5J and Suppl. Figures 2C,D were acquired with a Leica M205FA stereo microscope and display live fluorescence of fluorescent proteins. High resolution optical sections were obtained with a Leica SP8 confocal microscope or a Zeiss AxioObserver 7 equipped with an Apotome and processed using Fiji (Schindelin et al., 2012) (link).
Images in Figures 1C; 3A; 5B, G; Suppl. Figures 1A,B,E; Suppl. Figures 3C,D and movie S1 are z-projections. Figure 4A shows z-planes. Figure 1B contains an epifluorescence image obtained with a Zeiss AxioObserver 7. Movie S2 was recorded with a Leica M205FA stereo microscope. Movie annotations were added using the Annotate_movie plugin (Daetwyler, Modes and Fiolka, 2020) (link).
Images in Figures 1C; 3A; 5B, G; Suppl. Figures 1A,B,E; Suppl. Figures 3C,D and movie S1 are z-projections. Figure 4A shows z-planes. Figure 1B contains an epifluorescence image obtained with a Zeiss AxioObserver 7. Movie S2 was recorded with a Leica M205FA stereo microscope. Movie annotations were added using the Annotate_movie plugin (Daetwyler, Modes and Fiolka, 2020) (link).
6
Quantifying Viability of PC12 Cells
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PC12 cells were seeded on the 24-well plate at 1 × 104 cells. One to two days after seeding, the cells were treated with Pd(bpy) or Pd(sulfo-bpy) for 3-4 days. To visualize the live cells, the cells were loaded with 2 µM calcein-AM (dojindo) for 15 min in HBS, and then washed with HBS. The fluorescent images of the cells were obtained with fluorescent microscopy (Axio Observer7, Carl Zeiss) equipped with CMOS camera (Axiocam712mono, Carl Zeiss), and the cell numbers were automatically counted with ZEN blue software (Carl Zeiss).
7
Multicellular Microsystem Imaging Protocol
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In order to confirm the three-dimensional, spatial, and multilayered arrangement of cells grown in the microsystem, the non-malignant and cancer cells were stained with fluorescent dyes (CellTrackers, Thermo Fisher Scientific, Waltham, MA, USA). The non-malignant cells (HMF) were stained with red CMTPX dye. For this purpose, the cells in the culture vessel were incubated with 1 mL of CMTPX solution at a concentration of 6.25 µg/mL for 45 min (37 °C, 5% CO2). MCF-7 cells were stained with CMFDA, which showed green fluorescence. For this purpose, the cancer cells were incubated with 1 mL of CMFDA dye solution at a concentration of 5 µg/mL, also for 45 min (37 °C, 5% CO2). CellTracker dye working solutions were prepared via 1000-times dilution of the stock of CMFDA and CMTPX solutions in a culture medium without FBS and phenol red. The stained cells were detached from the surface, then introduced into a microsystem and cultured for 96 h. After incubation, a Z-axis scan of cell culture was performed using a confocal microscope (Zeiss Axio Observer 7 with LSM 900). Image acquisition was performed using ZEN Blue 3.6 software.
8
Immunofluorescence Analysis of Colonoid Proliferation
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For immunofluorescence staining, the colonoids were collected from Matrigel with Cell Recovery Solution (Corning, #354253) and fixed with 4% PFA for 30 min. The next steps were the same as performed in the tissue sections (described above). The BeyoClick™ EDU Cell Proliferation Kit with Alexa Fluor 594 (C0078S, Beyotime Biotechnology, Shanghai, China) was used to evaluate cell proliferation in colonoids. Briefly, the colonoids were treated with EDU (10 μM) at 37 °C for 2 h and then fixed in 4% PFA at room temperature for 15 min. After permeabilization with 0.3% Triton for 15 min, the colonoids were reacted with reaction mixture fluid for 30 min. The nuclei were stained with DAPI for 10 min. The images of the colonoids were captured using a fluorescence microscope (Axio Observer 7, Carl Zeiss). The ratio of positive cells was calculated as previously described.
9
Quantitative Microscopy of Bioaerosol Samples
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Imaging was done on a Zeiss Axio Observer 7 equipped with a 100× plan-apochromatic phase 3 oil immersion objective with a numerical aperture of 1·4. Epifluorescent illumination was provided by a 475 nm LED and non-specific fluorescence was removed with a Zeiss 38 HE filter set. Images were acquired using the Zeiss Zen software, and quantitative data extracted using MicrobeJ [38 (link)]. For serial imaging of bioaerosol samples, re-identification of putative bacilli detected at Day 0 was done by determining the x-y coordinates of the specific nanowell relative to the top-most, center nanowell in the macro well.
10
Cardiac Morphological Analysis Protocol
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For cardiac morphological analyses, hearts were embedded in OCT Tissue‐Tek and frozen at −80°C. Cardiac cryosections were stained with H&E as described.15 Interstitial collagen was analysed in picro‐Sirius red F3BA‐stained LV cryosections.15 Cardiomyocyte cross‐sectional area (CSA) was determined in longitudinal 6‐μm LV cryosections stained with rhodamine‐labelled wheat germ agglutinin (dilution 1:150; WGA, RL‐1022, Vector Laboratories) and Hoechst 33258 (Sigma‐Aldrich) for nuclear staining.17 At least a minimum of 50 representative cardiomyocytes (three sections/heart) were measured per heart. Inflammation was stained in LV cryosections with antibodies recognizing the monocyte–macrophage marker CD68 (dilution 1:1000; abcam ab53444) counterstained with WGA and Hoechst 33258 as described.18 Images were acquired by fluorescence and bright‐field microscopy using Axio Observer 7 and Zen 2.6 pro software (Carl Zeiss Jena).
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