Neurosphere assay was performed following a previously published protocol [23 ]. DG from each group of mice was collected and dissociated using the mixture of the enzymes containing 0.8 U/ml dispase (Stem cell, 07923), 2.85 U/ml papain (Worthington, LK003176) and 250 U/ml DNase (Stem cell, 07900) for 30 min at 37°C. Dissociated cells were plated in fresh DMEM/F12 with 100 ng/ml basic fibroblast growth factor and epidermal growth factor at a density of 1 × 104 cells per well of 96-well plate. Culture medium was added every 2 days during the entire culture period. To determine the NSC activity, the number and size of the spheres were determined 7 days after plating. Image of neurospheres were recorded using a digital camera attached to an Axiovert fluorescent microscope (Carl Zeiss) at a 20× magnification. Four images were randomly acquired from each plate, and the diameter of the neurospheres in these images was measured with a minimum cutoff diameter of 20 μm using the Image J software.
Axiovert fluorescent microscope
Manufactured by Zeiss
Sourced in Germany
The Axiovert fluorescent microscope is a laboratory equipment designed for high-resolution imaging of fluorescently-labeled samples. It provides a versatile platform for a wide range of applications, including cell biology, neuroscience, and materials science research. The Axiovert microscope utilizes advanced optics and illumination systems to enable detailed observation and analysis of fluorescent specimens.
Automatically generated - may contain errors
Lab products found in correlation
μ slide 6, by Ibidi (6 mentions)
Orca er digital camera, by Hamamatsu Photonics (6 mentions)
Vectashield mounting fluid with dapi, by Vector Laboratories (6 mentions)
Lsm 510, by Zeiss (6 mentions)
Triton x 100, by Merck Group (4 mentions)
Dnase, by STEMCELL (3 mentions)
Papain, by Worthington (3 mentions)
Dispase, by STEMCELL (3 mentions)
Vectashield hardset antifade mounting medium with phalloidin, by Vector Laboratories (3 mentions)
Axiocam mrm, by Zeiss (3 mentions)
Hoechst, by Thermo Fisher Scientific (3 mentions)
Anti flag m2 antibody, by Merck Group (3 mentions)
Alexa 488, by Jackson ImmunoResearch (3 mentions)
Duolink in situ mounting medium with dapi, by Merck Group (3 mentions)
Duolink in situ pla technology, by Merck Group (3 mentions)
Anti cd11c, by BD (3 mentions)
Anti cytokeratin 5 anti rabbit cy5, by BD (3 mentions)
Anti cytokeratin 8 anti rat cy3, by BD (3 mentions)
Mouse monoclonal anti brdu antibody, by Roche (3 mentions)
Alexa 488, by Thermo Fisher Scientific (3 mentions)
45 protocols using axiovert fluorescent microscope
1
Neural Stem Cell Sphere Assay
2
Cell Spreading on ECM Coated Surfaces
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HB2 and HB2 FGFR2(-) cells were seeded into fresh 60-mm cell culture dishes the day before an assay. Coverslips were coated with 100 µg/ml of freshly prepared Matrigel® Basement Membrane Matrix, rat tail Collagen type I or Fibronectin overnight at 4ºC. Coated coverslips were washed with PBS and blocked with 1 mg/ml BSA for 30 min. Next, cells were detached with enzyme-free cell dissociation buffer EDTA-based, seeded onto coated coverslips in 12-well plate and allowed to attach and spread for 90 min at 37ºC. After washing with PBS, cells were fixed in 4% paraformaldehyde (PFA) at RT, permeabilised with 0.1% Triton X-100 at 4 °C and mounted with Vectashield® HardSet™ Antifade Mounting Medium with Phalloidin (Vector Laboratories). The extent of cell spreading was quantified as an area of phalloidin signal per cell using ImageJ software. Representative images were taken using ZEISS AxioVert fluorescent microscope.
3
Autophagy Visualization with GFP-RFP-LC3
4
Autophagy Monitoring via GFP-RFP-LC3
5
Visualizing and Analyzing Polarized Cells
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Zeiss Axiovert fluorescent microscope equipped with an AxioCam MRm digital camera was used to visualize fluorescent and differential interference contrast microscopy (DIC) images. Axiovision (version 4.0) was used to capture the images that were further processed by Adobe Photoshop CS4 software. Percentage of the polarized cells was determined as described [13] (link).
6
Immunofluorescence Staining of FLAG-tagged Proteins
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Cells were grown on glass coverslips and fixed with flash treatment of ice-cold methanol before permeabilization with 0.25% Triton X-100 in phosphate-buffered saline for 5 min at room temperature. Slides were incubated with mouse monoclonal anti-FLAG M2 antibody (1:500; F1804; Sigma) for 2 h at 37 °C, followed by incubation with the appropriate secondary antibody coupled to Alexa-488 (1:1000; Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA). All cells were co-stained with Hoechst (33342; Invitrogen). Cells were mounted using Mowiol mounting medium and examined by fluorescence microscopy using an Axiovert fluorescent microscope (Zeiss).
7
Cell Viability Assessment of 3D Hydrogels
8
Live Cell Imaging of Autophagy
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Cells were transfected with a plasmid to express a green fluorescent protein (GFP) and red fluorescent protein (RFP) tagged form of LC3 (ATG8). For analysis of cells transfected with the GFP-RFP-LC3 construct, the GFP/RFP-positive vesicularized cells were examined under the ×40 objective of a Zeiss Axiovert fluorescent microscope.
9
Visualizing Autophagy with GFP-LC3
10
Polymer-Mediated Efficient Cellular Uptake and Endosomal Release of siRNA
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Example 17
This example demonstrates that polymer PRx0729v6 can mediate a more efficient cellular uptake of fluorescent-labeled siRNA and endosomal release than a lipid-based transfection reagent.
HeLa cells were plated on a Lab-Tek II chambered coverglass. Following overnight incubation, cells were transfected with either 100 nM FAM-siRNA/lipofectamine 2000 or with 100 nM FAM-siRNA at a Polymer-siRNA 4:1 charge ratio. Complexes were formed in PBS pH 7.4 for 30 minutes at a 5× concentration, added to cells for final 1× concentration, and incubated overnight. Cells were stained with DAPI (for visualization of the nucleus) for 10 minutes and then fixed in 3.7% formaldehyde-1×PBS for 5 minutes and washed with PBS. Samples were imaged with a Zeiss Axiovert fluorescent microscope.
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