Cells were rinsed with Phosphate Buffered Saline (PBS) and fixed with either 4% paraformaldehyde in PBS for 10 min at room temperature or with methanol for 10 min at 4°C. They were then permeabilized with 0.5% Triton-X 100 in PBS 3 times for 10 min. The same solution was used for all subsequent washing steps. Cells were incubated with primary antibodies for 1 h at 37°C. After incubation, cells were washed for 30 min and incubated with Alexa Fluor-conjugated secondary antibodies for 1 h at 37°C, and nuclei were labeled with DAPI (0.1 ηg/ml in 0.9% NaCl). Cells were mounted in Prolong Gold solution (Invitrogene) and examined with an Axiovert 100 microscope (Carl Zeiss, Germany) or with a laser scanning confocal microscope (TCS SP5 AOBS, Leica, Japan). Image processing and stack projections were performed using Fiji software (based on ImageJ, http://imageJ.nih.gov/ij/ ). Control experiments with no primary antibodies showed only a faint background staining (data not shown). Some cultured cells were also examined under phase contrast microscopy with an Axiovert 100 microscope (Carl Zeiss, Germany).
Axiovert 100 microscope
Manufactured by Zeiss
Sourced in Germany, United States
The Axiovert 100 is an inverted microscope designed for advanced imaging applications. It features a stable and vibration-free optical system, allowing for high-resolution observation and analysis. The Axiovert 100 is equipped with a range of observation techniques, including brightfield, phase contrast, and differential interference contrast, enabling a versatile and comprehensive analysis of samples.
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Lab products found in correlation
Dsu spinning disk confocal scanner, by Olympus (3 mentions)
Dafm 2x bioscope, by Veeco (3 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (3 mentions)
Dp71 digital camera, by Olympus (3 mentions)
Prolong gold, by Thermo Fisher Scientific (3 mentions)
Ixon3 emccd, by Oxford Instruments (2 mentions)
Igor pro, by Wavemetrics (2 mentions)
Inverted fluorescent microscope, by Olympus (2 mentions)
Lsm 510, by Zeiss (2 mentions)
Hpa002018, by Merck Group (2 mentions)
Fabppm, by Novus Biologicals (2 mentions)
Fatp1, by Novus Biologicals (2 mentions)
Fatp 4, by Abnova (2 mentions)
Wheat germ agglutinin alexa fluor 594 conjugate, by Thermo Fisher Scientific (2 mentions)
Lsm image browser, by Zeiss (2 mentions)
Cytosine arabinoside arac, by Merck Group (2 mentions)
Axiocam mrc5, by Zeiss (2 mentions)
Dp73 digital camera, by Olympus (2 mentions)
Transferman nk2, by Zeiss (2 mentions)
Genomeplex single cell whole genome amplification kit, by Merck Group (2 mentions)
71 protocols using axiovert 100 microscope
1
Immunofluorescence Staining Protocol
2
Senescence-associated β-galactosidase staining
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Following the treatments indicated in the Figure legends, subconfluent cells were washed three times in PBS, fixed with 4% parafomaldehyde (Santa Cruz Biotechnology, Dallas, USA) for 5 min, washed twice with PBS and incubated with fresh β-galactosidase staining solution (40 mM citric acid/sodium phosphate buffer pH 6, 5 mM K4[Fe(CN)6].3H2O, 5 mM K3[Fe(CN)6], 150 mM sodium chloride, 2 mM magnesium chloride and 1 mg mL−1 X-gal in distilled water) overnight at 37 °C in a humidified atmosphere. The following day, cells were washed twice in PBS, once in methanol and allowed to air dry. Images of SA-β-gal-stained cells were taken on a Zeiss Axiovert 100 microscope (Carl Zeiss Ltd, Cambridge, UK).
3
Quantitative Analysis of Prospore Membrane Formation
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Differential interference contrast (DIC) images and fluorescence images were obtained with a BX71 microscope (Olympus, Tokyo, Japan), a Quantix 1400 camera (Photometrics), and IPLab 3.7 software (Scanalytics).
Nuclear capture counting and prospore membrane perimeter measurement were performed as previously described (Okumura et al., 2016 ). Cells expressing both Htb2-mCherry and GFP-Spo2051–91 were sporulated for 9 h, and postmeiotic cells were identified by the pattern of Htb2-mCherry localization. The percentage of prospore membranes capturing nuclei is shown for each strain. Prospore membrane perimeters were measured by ImageJ (http://rsb.info.nih.gov/ij/ ).
Time-lapse imaging was performed as previously described (Ishihara et al., 2009 (link)). Images were captured on a Zeiss Axiovert 100 microscope (Carl Zeiss, Oberkochen, Germany) equipped with a CoolSNAP HQ camera (Photometrics) at 2-min intervals with IPLab 3.6.5a software (Scanalytics). The temperature was held at 28°C during image collection. Three-dimensional stacks were performed with IPLab 3.6.5a.
Nuclear capture counting and prospore membrane perimeter measurement were performed as previously described (Okumura et al., 2016 ). Cells expressing both Htb2-mCherry and GFP-Spo2051–91 were sporulated for 9 h, and postmeiotic cells were identified by the pattern of Htb2-mCherry localization. The percentage of prospore membranes capturing nuclei is shown for each strain. Prospore membrane perimeters were measured by ImageJ (
Time-lapse imaging was performed as previously described (Ishihara et al., 2009 (link)). Images were captured on a Zeiss Axiovert 100 microscope (Carl Zeiss, Oberkochen, Germany) equipped with a CoolSNAP HQ camera (Photometrics) at 2-min intervals with IPLab 3.6.5a software (Scanalytics). The temperature was held at 28°C during image collection. Three-dimensional stacks were performed with IPLab 3.6.5a.
4
Morphological Analysis of Effervescent Formulation
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EF-2 was placed on a brass stub with the application of double-sided adhesive tape, and then coated in a vacuum chamber with a thin layer of gold for 30 seconds (Sputter coater, Edwards, S150A, England, UK) to prepare it as electrically conductive. The pictures were taken at an excitation voltage of 20 kV (Electron probe microanalyzer, JEOL, JXA-840A, Tokyo, Japan) to check the surface morphology of the selected formula. A confocal laser scanning microscope was used to examine the surface morphology of EF-2. A Biorad MRC 1024 Laser Scanning Confocal Imaging System (Hemel Hempstead, UK), equipped with an argon ion laser (American Laser Corp, Salt Lake City, UT, USA) and a Zeiss Axiovert 100 microscope (Carl Zeiss, Oberkochen, Germany) was used to examine the effervescent formulation.
5
Quantifying Neuronal Process Length
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Micrographs of GAP-43-stained neurons were recorded using an inverted Zeiss Axiovert 100 microscope equipped with a 20x, 0.75 NA objective and coupled to a Zeiss, AxiocamMRm video camera (Carl Zeiss A/S, Birkeroed, Denmark). The average length of processes per neuron was estimated using a stereological approach as described50 (link). For each well, 150–200 neurons were captured and analyzed.
6
Wound Healing Assay for Cell Migration
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To assess the ability of cells to move, the wound healing experiment was performed.MDA-MB-231 cells were plated in 24-well plates with 5 × 104 cells per well. Then, the tip of a 200 µL tip was used to remove the cells in a uniform manner. Each well was washed 3 times with PBS and then three wells received treatments with 1 µg/mL of DTX and SLN-DTX or only the culture medium with 1% fetal bovine serum, as a control. After 24 h, 48 h, and 72 h, the images of each treatment were recorded on an Axiovert 100 Microscope (Zeiss, Germany). For image analysis, ImageJ Software was used to calculate the area without cells, and each time three images were used for each treatment.
7
Microfluidic Bending Rigidity of E. coli
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The bending rigidity of E. coli cells was measured using a microfluidic assay similar to that used in Refs. 20 (link),21 (link). E. coli MG1655 was transformed with a plasmid (pDB192) containing sulA under an isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible promoter. Cells were grown overnight in 2 mL LB containing 30 μg/mL kanamycin and 50 μg/mL ampicillin. IPTG was added to the medium to induce sulA throughout cell growth in the microfluidic flow chamber. Deflection of cells under fluid flow was monitored on a Zeiss Axiovert 100 microscope (Zeiss) equipped with a 60X oil objective. Images were collected with an Andor iXon 3 EMCCD (Andor) using μManager v. 1.426 . Deflection of the cells was determined using a custom Igor Pro (WaveMetrics Inc.) image-analysis algorithm.
8
Microscopy Imaging and Processing Protocol
9
Affinity Purification and Fibroblast Assay
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mAb NZ-1, described above, was affinity purified using a Protein G HP spin trap column (GE Healthcare, Cat.#28903134) and concentrated using an ultra-centrifugal filter (EMD Millipore, Cat.# UFC905024) [27 (link)]. The concentration was measured by a Nanodrop 1000 spectrophotometer (ThermoScientific) and 0.67 μg added to 4x104 F-NHDFs or CC-NHDFs in 200 μl of medium. The preparations were incubated at room temperature for 20 minutes. The contents of each tube were then divided equally between two collagen-coated wells. As controls, mAb in 200 μl PBS was processed similarly in the absence of cells. The plates were then incubated for 30 minutes at 5% CO2 at 37°C. An MDA-MB-231/Matrigel mixture was then cast over the fibroblast cell layer as described above. Images were acquired daily for four days using the 10X objective of a Zeiss Axiovert 100 microscope.
10
Quantifying Oligodendrocyte Morphology
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WT and TMEM10 Oli-sneu cells were cultured under proliferation conditions for 48 hrs. Cells were then fixed with 4% PFA and stained with Cell Tracker dye (ThermoFisher). Images were taken with an ImageXpress microscope (Molecular Devices) and analyzed using MetaXpress software. Measurements taken included process outgrowth; number of branches per cell; number of processes per cell; and percentage of cells exhibiting process outgrowth. For morphological analysis of primary OPCs, cell cultures were fixed in 4% PFA and immunolabeled for MBP. Images were taken using an Axiovert 100 microscope (Carl Zeiss) with a MagnaFire CCD camera (Optronics). 50–100 cells were randomly selected and individually outlined using the selection tool in ImageJ41 (link). Mean fluorescence intensity and cell area were measured and multiple shape descriptors calculated using ImageJ. Circularity is defined as the degree to which a particle is similar to a circle, taking into consideration the smoothness of the perimeter and solidity as an inverse measure of the overall concavity of a particle42 .
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