Bacillus strains were grown in LB medium to the mid-log or stat phase. For cell length measurements, at each time point 500 µl of cells was harvested, and the cells were incubated for 5 min in 2 µg/ml propidium iodide (PI) on poly-l -lysine-coated slides, washed 10× with phosphate-buffered saline (PBS; 81 mM Na2HPO4 plus 24.6 mM NaH2PO4 plus 100 mM NaCl), and then mounted in Slowfade (Molecular Probes). For wheat germ agglutinin (WGA [Life Technologies]) staining, cells were attached to a poly-l -lysine-coated slide, incubated for 1 min in the presence of 2 mg/ml (final concentration) lysozyme, and then immediately washed three times in PBS. Cells were stained for 15 min with 2 µg/ml (final concentration) 4′,6-diamidino-2-phenylindole (DAPI) and 1 µg/ml WGA and then washed and mounted as described above. Microscopy was performed with an upright Nikon Eclipse 90i microscope outfitted with an Orca-ER digital camera (Hamamatsu) and a Nikon TIRF 1.45 NA Plan Neo-Fluor 100× oil immersion objective. The Volocity software package (version 6.3; PerkinElmer) was used for image acquisition and measurements. The exposure time for the WGA staining was 50 ms for every strain in both growth phases, with the exception of the mreB mutant in log phase, which was imaged for 10 ms. For individual cell measurements, the length and width of 150 cells were determined for each strain.
Eclipse 90i
Manufactured by Hamamatsu Photonics
Sourced in United States
The Eclipse 90i is a high-performance, versatile laboratory instrument designed for a wide range of scientific applications. It features a compact and modular design, allowing for customization to meet the specific needs of researchers and scientists. The Eclipse 90i is capable of performing various analytical tasks, including spectroscopy, imaging, and detection, making it a valuable tool for a diverse range of research disciplines.
Automatically generated - may contain errors
Lab products found in correlation
Orca er, by Universal Imaging (4 mentions)
Metamorph, by Universal Imaging (4 mentions)
Orca er, by Hamamatsu Photonics (2 mentions)
Wheat germ agglutinin (wga), by Thermo Fisher Scientific (1 mentions)
Slowfade, by Thermo Fisher Scientific (1 mentions)
Volocity software package, by PerkinElmer (1 mentions)
C10600 camera, by Hamamatsu Photonics (1 mentions)
Immpress reagent kit, by Vector Laboratories (1 mentions)
Eclipse e800, by Nikon (1 mentions)
Dab peroxidase substrate kit, by Vector Laboratories (1 mentions)
7 protocols using eclipse 90i
1
Bacillus Cell Morphology Characterization
2
Rhodamine-labeled pSVCTQ Liver Distribution
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Rhodamine-labeled pSVCTQ was formulated in PBS and SC administered in mice at a dose of 25 mg/kg TQ equivalent. Mice receiving PBS served as controls. The liver was harvested at 8 hours after injection and immediately fixed in 10% neutral buffered formalin at 4°C for 4 hours. After washing three times in cold PBS, the liver tissue was sunk in 15% sucrose solution for 24 hours and then in 30% sucrose solution for 24 hours at 4°C. The sucrose solution was prepared in DPBS (1×, with Mg2+ and Ca2+). The liver tissue was then blotted with paper tissue and embedded in an optimal cutting temperature compound. The sample was frozen in prechilled isopentane in liquid nitrogen and stored at −80°C. The 5-μm-thick sections were prepared using CryoStat at −20°C. Alexa Fluor 488 phalloidin was diluted 1:50 in PBS and used to counterstain the sections for 1 hour at room temperature (RT) to visualize cell outlines. The cell nuclei were counterstained with DAPI (blue) for 5 min at RT. Sections were imaged using an epi-fluorescent microscope (Nikon Eclipse 90i and Hamamatsu C10600 Camera at 20× magnification) using GFP-B HYQ (excitation/emission: 450/550 nm) Texas red HYQ (excitation/emission: 535/580 nm) and ultraviolet illumination (excitation/emission: 350/470 nm).
3
Quantifying Hypoxia and Signaling in 3D Spheroids
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To quantify hypoxia, spheroids were incubated with 200 μM EF5 (2-(2-Nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl) acetamide), a gift from Cameron Koch’s lab) for 6 h at 37 °C, prior to overnight fixation in 4% pFA in PBS and preservation in 30% sucrose in PBS at 4 °C. 8 μm sections were incubated overnight at 4 °C with the EF5 Cy3-conjugated monoclonal antibody ELK3-51 (www.hypoxia-imaging.org ) and counterstained with Hoechst 33342. Images were acquired with a Nikon Eclipse 90i with a Hamamatsu ORCA-ER camera. A mask was created for each spheroid using the Hoechst image, prior to measuring the average pixel intensity per spheroid in the background-subtracted EF5 image using ImageJ [1] .
To assess signaling inhibition in spheroids, sections were stained with anti-pAKT antibody using ImmPRESS™ reagent kit (MP-7401, VectorLabs) and DAB Peroxidase substrate kit (SK-4100, VectorLabs). Images acquired with a Nikon Eclipse E800 were analyzed for 3,3′-Diaminobenzidine (DAB)-positive pixels semi-automatically using a method described previously [6] (link).
To assess signaling inhibition in spheroids, sections were stained with anti-pAKT antibody using ImmPRESS™ reagent kit (MP-7401, VectorLabs) and DAB Peroxidase substrate kit (SK-4100, VectorLabs). Images acquired with a Nikon Eclipse E800 were analyzed for 3,3′-Diaminobenzidine (DAB)-positive pixels semi-automatically using a method described previously [6] (link).
4
Detecting Nuclear Localization of Cdc25
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Images were obtained using a Nikon Eclipse 90i fluorescence microscope with a Hamamatsu Orca-ER camera driven by Metamorph (Universal Imaging, Downingtown, PA). Images were further processed with ImageJ software.
To determine the presence of Cdc25 at nucleus, merged (RGB) images from cells carrying a cut11-cherry allele (marking the nuclear membrane) and a GFP-Cdc25 allele were used. In each nucleus, a line was traced covering all nucleus diameter and also surrounding cytoplasm using the ImageJ software and the plot intensity for each channel was obtained (using the RGB line profile plugging from ImageJ). Those cells in which the signal inside the nucleus was above the cytoplasmic background were considered as positive for nuclear GFP. In the few cases of strong cytoplasmic background, the sorting or not of that particular nucleus was decided case by case, by looking the intensity of nuclear signal.
To determine the presence of Cdc25 at nucleus, merged (RGB) images from cells carrying a cut11-cherry allele (marking the nuclear membrane) and a GFP-Cdc25 allele were used. In each nucleus, a line was traced covering all nucleus diameter and also surrounding cytoplasm using the ImageJ software and the plot intensity for each channel was obtained (using the RGB line profile plugging from ImageJ). Those cells in which the signal inside the nucleus was above the cytoplasmic background were considered as positive for nuclear GFP. In the few cases of strong cytoplasmic background, the sorting or not of that particular nucleus was decided case by case, by looking the intensity of nuclear signal.
5
Fluorescence Microscopy Imaging Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Images were obtained using a Nikon Eclipse 90i fluorescence microscope with a Hamamatsu Orca-ER camera driven by Metamorph (Universal Imaging, Downingtown, PA, USA). Images were further processed with Adobe Photoshop CS software.
6
Fluorescence Microscopy Image Processing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Images were obtained using a Nikon Eclipse 90i fluorescence microscope with a Hamamatsu Orca-ER camera driven by Metamorph (Universal Imaging, Downingtown, PA, USA). Images were further processed with ImageJ software.
7
Fluorescence Imaging with Nikon Eclipse 90i
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Images were obtained using a Nikon Eclipse 90i fluorescence microscope with a Hamamatsu Orca-ER camera driven by Metamorph (Universal Imaging, Downingtown, PA). Images were further processed with ImageJ software.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!