For coculture with Sema5A-Fc-expressing COS cells, 3 μL of transfected COS cells suspended in Matrigel were deposited in the center of the culture well 3h after the plating of gut dissociated cells. To assess the activity of full-length Sema5A on enteric neuron development and allow for cell-cell contact between enteric neurons and COS cells, gut dissociated cells were plated in a culture well containing transfected COS cells (400,000 cells/well). For these two procedures, cells were fixed 3 days after plating. Quantifications in the ENS-COS cell coculture were conducted in the vicinity of the Matrigel dot, within a range covering 0 to 900 μm from the Matrigel’s periphery, utilizing the Axio Observer Zeiss microscope and Zen software (Zeiss, Germany), with a x10 objective. The number of Hu-immunoreactive neurons per ganglion in ENS cultures was scored on 20 ganglia from 4 experiments using Axio Observer Zeiss microscope.
Axio observer microscope
Manufactured by Zeiss
Sourced in Germany, Canada, United States, France
The Axio Observer is a high-performance inverted microscope from Zeiss. It is designed for a wide range of applications, including cell culture observation, live-cell imaging, and high-resolution imaging. The microscope features a stable and ergonomic design, allowing for precise and reliable performance. It is equipped with advanced optics and illumination systems to provide clear and detailed images.
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Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (16 mentions)
Hoechst 33342, by Thermo Fisher Scientific (15 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (13 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (11 mentions)
Lsm 880, by Zeiss (11 mentions)
Zen software, by Zeiss (10 mentions)
Apotome imaging system, by Zeiss (10 mentions)
Zen blue software, by Zeiss (8 mentions)
Axiocam, by Zeiss (7 mentions)
Triton x 100, by Merck Group (7 mentions)
Csu xi spinning disk confocal scan head, by Yokogawa (7 mentions)
Goat serum, by Thermo Fisher Scientific (6 mentions)
Axiovision software, by Zeiss (6 mentions)
Zen imaging software, by Zeiss (6 mentions)
Lsm 780, by Zeiss (6 mentions)
Lsm 700, by Zeiss (5 mentions)
Metamorph software, by Molecular Devices (5 mentions)
Zen 2012, by Zeiss (5 mentions)
Apotome 2, by Zeiss (5 mentions)
Axiocam mrm camera, by Zeiss (5 mentions)
539 protocols using axio observer microscope
1
Sema5A-Fc Co-culture Assay for Enteric Neurons
2
Osteogenic Differentiation Monitoring in MC3T3-E1 Cells
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MC3T3-E1 cells (8 × 105 cells per well) were cultured in the co-stimulation media consisting of an osteogenic induction medium and the above-mentioned macrophage-conditioned media with different treatments at a ratio of 2:1. Alkaline phosphatase assay (ALP) was performed as early osteogenic evaluation at day 7. In brief, the cells were incubated in 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium (BCIP/NBT; Beyotime) for 30 min, following fixation in 4% PFA for 30 min at room temperature. ALP-positive cells were observed under an Axio Observer microscope (Carl Zeiss, Oberkochen, Germany). For the quantitative analysis of ALP, the cells were lysed in 1% Triton X-100 solution for 1 h, and the supernatant was taken after low-speed centrifugation (1500 rpm, 5min). The supernatant was detected by the Alkaline phosphatase kit (Jiancheng, China) according to the instructions. The absorbance was determined at 520 nm using a microplate reader (Biotek, Germany). Calcium accumulation, the late osteogenic marker, was assessed by Alizarin Red Staining after 21 d of above-mentioned co-stimulation. Cells were fixed in 4% PFA for 30 min, and then were stained with 40 mM Alizarin Red (pH 4.2; Cyagen, China) for another 30 min. After three washes with deionized water, ALP-positive cells were observed under the Zeiss Axio Observer microscope.
3
Quantitative Analysis of Phosphorylated Tau
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The light microscopy images during the culture (live images DIV0-75) were obtained with a Leica MZ6 microscope (0.5× objective) equipped with the MC120 camera (Leica Microsystems GmbH, Wetzlar, Germany). Fluorescence wide-field images were obtained with a Leica DM6B-Z microscope with a K5-14400955 camera and Thunder image processing (Leica Microsystems GmbH). Confocal fluorescence images were obtained with a Zeiss Axio Observer microscope equipped with the LSM700 or LSM800 confocal module (Carl Zeiss Microimaging GmbH, Jena, Germany). The light, laser, and detector settings were kept constant for all samples for each specific immunostaining. Quantitative image analysis was carried out using the Fiji ( ImageJ 1.53c) software [20 (link)]. The Z-stacks from each channel were maximum-intensity projected, background subtracted (using the rolling ball algorithm), and Gaussian blurred. To quantify the phosphorylated TAU for experiment 1, the pTAU and DAPI channels were thresholded with the Moments threshold (pTAU 5907/65535, DAPI 11447/65535), and the regions of interest (ROI) selected were with the Analyze Particles function. For experiment 2, the pTAU channel was thresholded with the Triangle automatic threshold, the DAPI channel with the Li automatic threshold, and segmented with Watershed prior to the Analyze Particles function.
4
Cell Migration Tracking Assay
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Cells were seeded in 35 mm Petri dishes and cultured until confluence. The cells were then scraped with a 200 μl micropipette tip, denuding a strip of the monolayer and placed onto a Zeiss Axio Observer Microscope (Zeiss, Germany) equipped with an incubation chamber (ibidi Heating System, Ibidi, Germany) set at 37 oC. Phase contrast images were taken every 20 min during 48 h using a camera AxioCam HRm from Zeiss. The movies were processed using the cell tracking plug-in from Fiji (http://fiji.sc/Fiji ) to extract cell trajectories and analyzed to obtain the MSD following equation 1 , where x and y are the coordinates of the particle, t is time, and the brackets represent the trajectories ensamble average (see equation 1 ).
5
Generating Akata/FNF-DsRed Reporter Cells
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For generating reporter cells, Akata(−) cells were inoculated with the lentiviral vector harboring the CMV promoter-driven FNF-DsRed cassette and followed by G418 selection (750 μg/mL).
Akata/FNF-DsRed cells were incubated in medium containing EBV(BMRF1p-Flpe) at room temperature for 2 h with agitation. The cells were spun down, resuspended in fresh medium, and then cultured. Expressions of fluorescent proteins, EGFP, and DsRed were observed at discrete time points. Images were acquired using a Zeiss Axio Observer microscope (Carl Zeiss, Oberkochen, Germany).
Akata/FNF-DsRed cells were incubated in medium containing EBV(BMRF1p-Flpe) at room temperature for 2 h with agitation. The cells were spun down, resuspended in fresh medium, and then cultured. Expressions of fluorescent proteins, EGFP, and DsRed were observed at discrete time points. Images were acquired using a Zeiss Axio Observer microscope (Carl Zeiss, Oberkochen, Germany).
6
Immunofluorescent Staining of Murine Liver
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Cryo-sectioned 5 μm liver tissue slides were brought to room temperature and fixed with cold acetone for 8 min and then washed in PBS containing 0.05% Tween 20 (PBS-T). Nonspecific reactions were blocked with 5% normal goat serum (ab7481, Abcam, Cambridge, MA) and 5% TruStain fcX (BioLegend, San Diego, CA) in PBS-T for 1 h and then incubated with rabbit anti-mouse F4/80 (1:200, ab111101, Abcam) and rat anti-mouse Ly-6C (1:200, ab24973, Abcam) at 4°C overnight. After washing in PBS-T, the specimens were incubated with Alexa Fluor 488 goat anti-rat IgG (H+L) (1:400, #4416, Cell Signaling, Danvers, MA) and Alexa Fluor 555 goat anti-rabbit (1:400 #4413, Cell Signaling) for 1 hour at room temperature, washed again, treated with Vector TrueVIEW Autofluorescence Quenching Reagent and then counterstained with VECTASHIELD Mounting Medium with 4’,6-diamino-2-phenylindole (DAPI) (both: Vector Labs, Burlingame, CA). The Zeiss Axio Observer Microscope (Carl Zeiss Micro Imaging, Inc., Thornwood, NY) equipped with the Zen pro 2.3 software was used to visualize the immunofluorescence staining for F4/80 and Ly-6C, and nuclear localization was provided by DAPI. The negative controls were obtained by incubating sections with non-specific rat IgG or rabbit IgG as described above.
7
Biocompatibility and Cell Attachment Evaluation
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Biocompatibility and cell attachment to the mesh were evaluated by seeding HDFs (10,000 cells/well) onto ethanol-sterilised mesh samples (15.6 mm diameter). The seeding efficiency of LCM1 and LCM2 was 10 and 5%, respectively (data not shown). Samples were analysed at 3, 7, 14 days.
Proliferation of HDFs seeded on the meshes was evaluated with alamarBlue (Invitrogen, Thermo Fisher), with 1:9 ratio (alamarBlue to growing media ratio). Control samples were HDFs cultured in DMEM supplemented with FBS and P/S. Samples were incubated for 4 h at 37 °C, 5% CO2 and transferred to a 96-well plate (triplicates of 100 µl from each sample). Samples were imaged with a CLARIOstar® microplate reader (BMG LABTECH GmbH, Germany) in fluorescence mode (excitation 560 nm, emission 590 nm).
Samples of the seeded meshes were fixated in 4% paraformaldehyde for 30 min and washed with Phosphate Buffer Solution (Thermo Fisher Scientific). After permeabilization using 0.25% Triton X-100 (Sigma Aldrich), samples were washed in PBS and stained using Phalloidin TRITC (Sigma) 1:1000 in PBS for 1 h to visualise cells’ actin filaments. Following these steps, the samples were mounted on glass microscope slides with VECTASHIELD® DAPI (Vector Laboratories Inc.) to visualise nuclei. Samples were imaged with Zeiss AxioObserver Microscope with ApoTome.2 feature and Zeiss ZEN software (Zeiss, Oberkochen, Germany).
Proliferation of HDFs seeded on the meshes was evaluated with alamarBlue (Invitrogen, Thermo Fisher), with 1:9 ratio (alamarBlue to growing media ratio). Control samples were HDFs cultured in DMEM supplemented with FBS and P/S. Samples were incubated for 4 h at 37 °C, 5% CO2 and transferred to a 96-well plate (triplicates of 100 µl from each sample). Samples were imaged with a CLARIOstar® microplate reader (BMG LABTECH GmbH, Germany) in fluorescence mode (excitation 560 nm, emission 590 nm).
Samples of the seeded meshes were fixated in 4% paraformaldehyde for 30 min and washed with Phosphate Buffer Solution (Thermo Fisher Scientific). After permeabilization using 0.25% Triton X-100 (Sigma Aldrich), samples were washed in PBS and stained using Phalloidin TRITC (Sigma) 1:1000 in PBS for 1 h to visualise cells’ actin filaments. Following these steps, the samples were mounted on glass microscope slides with VECTASHIELD® DAPI (Vector Laboratories Inc.) to visualise nuclei. Samples were imaged with Zeiss AxioObserver Microscope with ApoTome.2 feature and Zeiss ZEN software (Zeiss, Oberkochen, Germany).
8
Immunofluorescence Analysis of IL23R
9
Phase-Contrast Microscopy Image Analysis
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Phase-contrast images were acquired using a Zeiss Axio Observer Microscope (Carl Zeiss Canada, North York, ON, Canada) and analyzed using Zen 2 (Blue Edition) software (Carl Zeiss Canada). Images were processed using GNU Image Manipulation Program (GIMP)73 and compiled using Inkscape software.74
10
Immunofluorescent Staining of WT1 in Paraffin Sections
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For immunofluorescent stainings, 2 μm paraffin sections were deparaffinized and antigen retrieval was performed by boiling at 98°C in 0.05% citraconic acid anhydride (Sigma, St. Louis, USA), pH7.4 for 20 min. Unspecific binding was blocked in 3% goat serum for 60 min at RT. Primary antibody incubation (rabbit α-WT1 1:800, Santa-Cruz, Santa-Cruz, USA) was performed in blocking buffer o/n at 4°C. Binding was visualized by incubation with TRITC-α-rabbit coupled secondary antibodies (Abcam, Eugene, USA) diluted 1:300 in blocking buffer for 60 min at RT. Stainings were evaluated with a Zeiss Axio Observer microscope using the LSM software (Zeiss, Jena, Germany).
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