TRAP5 staining: Cell morphology and TRAP5-staining were imaged using bright field microscopy (Axiovert, Zeiss, Germany). The formation of tartrate resistant acid phosphatase (TRAP5)-positive cells was determined by using a commercially available kit (Sigma, Germany). Cells were fixed and stained after 14 days of cultivation, then examined using light microscopy (Axiovert, Zeiss, Germany), where TRAP-positive cells were a maroon color. Mineralization Assay (Lonza, Germany), where the stock solution was diluted 1:100 (v/v) and then subsequently incubated for 30 minutes at room temperature. Samples were washed and stored in 1x PBS until microscopic imaging. 3D confocal microscopic images were obtained and computer processed (Amira 3D Software for Life Sciences, FEI, USA) to build 3D schematic images.
Axiovert
Manufactured by Zeiss
Sourced in Germany, United States, United Kingdom, Italy
The Axiovert is a laboratory microscope designed for inverted microscopy. It provides a stable and reliable platform for a variety of applications, including cell culture observation, live-cell imaging, and developmental biology research. The Axiovert features high-quality optics and a modular design, allowing for customization to meet the specific needs of the user.
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Lab products found in correlation
Axiovision software, by Zeiss (9 mentions)
Lsm 510, by Zeiss (6 mentions)
Lsm 780, by Zeiss (5 mentions)
Pdgf bb, by R&D Systems (4 mentions)
Trypsin, by Thermo Fisher Scientific (4 mentions)
Amphotericin b, by Thermo Fisher Scientific (4 mentions)
Axiocam mrc, by Zeiss (4 mentions)
Axioplan microscope, by Zeiss (4 mentions)
Lsm 710, by Zeiss (4 mentions)
Triton x 100, by Merck Group (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
Matrigel, by BD (3 mentions)
Axiocam, by Zeiss (3 mentions)
Ccd camera, by Olympus (3 mentions)
Metafluor software, by Universal Imaging (3 mentions)
Lsm520 meta, by Zeiss (3 mentions)
Oil red o, by Merck Group (3 mentions)
Live dead viability cytotoxicity kit for mammalian cells, by Thermo Fisher Scientific (3 mentions)
Mitotracker red, by Thermo Fisher Scientific (3 mentions)
Ds qi2, by Nikon (2 mentions)
339 protocols using axiovert
1
Osteoclast Differentiation and Mineralization
2
Immunofluorescence Staining of Placental Cells
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The cells were cultured in a μ-Slide 8 Well chamber (ibidi) for 2 d (TSLCs and TSCs) or 8 d (EVT), and then fixed with 4% paraformaldehyde (PFA; Sigma-Aldrich) for 20 min at room temperature (RT). The ST were cultured for 6 d in Petri dish and collected into 1.5 mL tube. The ST were fixed with 4% PFA for 1 h at RT. The fixed cells were permeabilized and blocked with 0.3% Triton X-100 (Sigma-Aldrich) and 10% normal goat serum (Sigma-Aldrich) in Dulbecco’s phosphate-buffered saline (PBS; Thermo Fisher Scientific) for 45 min at RT. For HLA-G staining, a blocking solution was used without 0.3% Triton X-100. The samples were incubated with diluted primary antibodies (SI Appendix, Table S2 ) at 4 °C overnight. The cells were incubated with Alexa Fluor 488- or Alexa Fluor 594-conjugated goat secondary antibodies (1:400; Invitrogen) for 1.5 h at RT. The nuclei of the cells were counterstained with 4’,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich) for 5 min and the images were acquired using a fluorescence microscope (Zeiss Axiovert, Carl Zeiss).
3
Immunohistochemical Analysis of Neuronal Markers
4
Amphid Commissure Axon Phenotyping
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Animals are collected from plates in M9 with 10 μg/ml of lipophilic dye 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI, prepared in N,N-dimethylformamide), incubated in dye for 30 min in the dark, washed twice with M9, placed on bacteria plates for 3 h. Amphid-commissure axon phenotypes are scored in epifluorescent microscope Zeiss Axiovert (63×/1.4 NA objective, Zeiss Filter set 43 HE Cy3). Different defects in amphid commissures are scored as described below.
5
Quantitative Fluorescence Imaging Protocols
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Specimens were imaged using a 40× Zeiss Plan-Apochromat objective with 0.95 numerical aperture on a Zeiss Axiovert (Zeiss) epifluorescent microscope. All images were acquired with a CCD Hamamatsu Photonics (Herrsching am Ammersee, Germany) monochrome camera at 1392 × 1040 pixel size, 12 bits per pixel depth, and assembled as false-color images using the Metamorph imaging platform (Molecular Devices, Inc.).
For quantitation of cells immunoreactive for p-Smad2, three to five nonoverlapping images were obtained per section and analyzed using in-house algorithms in Fiji-Image J (NIH, Bethesda, MA). Nuclear masks based on DAPI served to determine the regions of interest (ROIs). The fraction of p-Smad2–positive pixels in each ROI was calculated. A threshold value that discriminates between positive and negative cells was determined using all ROIs. The nuclear immunofluorescence intensity of Sox2 was similarly quantified. γ-H2AX foci were enumerated using an algorithm similar to that previously described [38] (link). The mean number of γ-H2AX foci as a function of treatment was determined only from positive cells (i.e., nuclei with one or more foci). To quantitate nuclear size after irradiation, we used SigmaScan Pro 5 (Systat Software Inc.) to measure dimensions of 524 nuclei defined by ROIs.
For quantitation of cells immunoreactive for p-Smad2, three to five nonoverlapping images were obtained per section and analyzed using in-house algorithms in Fiji-Image J (NIH, Bethesda, MA). Nuclear masks based on DAPI served to determine the regions of interest (ROIs). The fraction of p-Smad2–positive pixels in each ROI was calculated. A threshold value that discriminates between positive and negative cells was determined using all ROIs. The nuclear immunofluorescence intensity of Sox2 was similarly quantified. γ-H2AX foci were enumerated using an algorithm similar to that previously described [38] (link). The mean number of γ-H2AX foci as a function of treatment was determined only from positive cells (i.e., nuclei with one or more foci). To quantitate nuclear size after irradiation, we used SigmaScan Pro 5 (Systat Software Inc.) to measure dimensions of 524 nuclei defined by ROIs.
6
Visualizing VACV Infection Dynamics
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BSC40 cells infected with VACV were imaged from 24-48 hpi in an environmental chamber maintained at 37°C and 5% CO2. Two different time-lapse microscope settings were used: A Zeiss Axiovert 200M with a Hamamatsu Orca AG camera, and a Nikon Ti inverted microscope with a Nikon DS-Qi2 high sensitivity scientific CMOS camera. A 4x objective was used for the Zeiss Axiovert, and a 10x objective was used for the Nikon Ti. For each condition, 10-15 plaques were imaged every 10 min. For plaques next to wounds, each well was scratched at 22 hpi with a 200 μl pipette tip, and the well was washed 2x with DMEM(-) before imaging as above.
7
Determining Cell Volume via CFDA
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Equilibrium cell volumes (Vo) were obtained after loading the cells with CFDA under an epifluorescent microscope (Zeiss Axiovert, Zeiss, Jena, Germany) equipped with a digital camera. Cells were assumed to have a spherical shape with a diameter calculated as the average of the maximum and minimum dimensions of each cell.
8
Fluorescent Bacterial Imaging Protocol
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Fluorescence microscopy samples were prepared from overnight bacterial cultures in LB broth. Samples were allowed to dry on l -lysine-coated coverslips, fixed with paraformaldehyde, and then poststained with the thiol-reactive dye Alexa Fluor 594 (Life Technologies, Paisley, UK). Residual fluorescent dye was removed by washing cells with phosphate-buffered saline (PBS) and samples were mounted onto glass microscope slides with ProLong Antifade Gold with DAPI (Life Technologies). Samples were analyzed on a Zeiss Axiovert (Zeiss, Cambridge, UK) fluorescence microscope at 1000× magnification.
9
PKC Activity Monitoring in CHO-K1 Cells
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CHO-K1 cells co-transfected with mCherry-PKCα and CKAR2 (C Kinase Activity Reporter) were maintained in HBSS solution (ThermoFischer) during the experiment. 1 ml of LB broth was added to settle a baseline. After 5 minutes S. aureus culture supernatants were added and, after 10 minutes, 1 μM of the PKC inhibitor Gö6976 was added. To study the effect of α-hemolysin, 10 μg/ml or 30 μg/ml of pure protein was added to the cells, followed by 200 nM of the PKC activator PDBu and finally 1 μM Gö6976. Images were acquired using the microscope Zeiss Axiovert (CarlZeiss Microimaging, Inc.) with the digital camera MicroMax (Roper-Princeton Instruments) controlled by the software Metafluor (Universal Imaging, Corp.). FRET (Föster Resonance Energy Transfer) and CFP (Cyan Fluorescent Protein) were obtained every 25 seconds. YFP (Yellow Fluorescent Protein) and mCherry emissions were also obtained as transfection controls.
10
Fluorescence Immunocytochemistry of Vascular Cells
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