Mitotic cells were identified by brighter DAPI fluorescence intensity compared to surrounding interphase cells, DNA morphology, and centrosome position. Prometaphase cells were identified by the wheel-like rosette organization of the chromosomes as described (Bolzer et al. 2005 (link); Magidson et al. 2011 (link); Itoh et al. 2018 (link); Nagele et al. 1995 (link)). Metaphase cells were identified by chromosome alignment at the equatorial plate, with a centrosome on either side. Fixed mitotic HUVECs were imaged with a confocal microscope (Leica TCS SPE, DM2500) using a 63x/1.3NA oil immersion objective with a digital zoom of 1.5x. The image data were acquired sequentially in a four-channel mode. Z-stacks were captured using a frame size of 1,024 ×1,024 pixels, and processed with Leica Application Suite X software (Version: 3.5.2.18963). Confocal optical sections were reconstructed and visualized in Imaris software (Bitplane: 9.8.2). Imaris deconvolution algorithm (iterations: 10, pre-sharpening gain: 7.0) was applied, and the fluorescence level for all channels was thresholded by including 90% of each signal.
Tcs spe dm2500
Manufactured by Leica
Sourced in Germany
The TCS SPE DM2500 is a confocal laser scanning microscope designed for high-resolution imaging. It features a laser source, scanning unit, and detection system to capture detailed images of samples. The core function of this product is to provide advanced microscopic imaging capabilities.
Automatically generated - may contain errors
Lab products found in correlation
Las af software, by Leica (2 mentions)
Dxm1200, by Nikon (2 mentions)
Eclipse e800, by Nikon (2 mentions)
Act 1, by Nikon (2 mentions)
Application suite x software, by Leica (1 mentions)
Tetramethylrhodamine fluorescent dye, by Merck Group (1 mentions)
Hoechst 33258, by Merck Group (1 mentions)
Hoechst 33258 cell nucleus dye, by Merck Group (1 mentions)
Texas red c2 maleimide cell membrane dye, by Thermo Fisher Scientific (1 mentions)
Nis element, by Nikon (1 mentions)
Imagej, by Nikon (1 mentions)
6 protocols using tcs spe dm2500
1
Mitotic Cell Imaging and Analysis
2
Visualizing Cell Cytoskeleton and Nuclei
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The spreading and the shape of the cells were evaluated by staining the cell F-actin cytoskeleton and the cell nuclei. The F-actin was stained with phalloidin conjugated with tetramethylrhodamine fluorescent dye (Sigma-Aldrich Co), and the cell nuclei were stained with Hoechst #33258 (Sigma-Aldrich Co) for 1 hour at room temperature in the dark. Both dyes were diluted in PBS to concentration 5 µg/mL. Before the cells were stained with fluorescent dyes, they were rinsed in PBS and were fixed with −20°C cold ethanol for 10 minutes. The images were taken using a Leica TCS SPE DM2500 upright confocal microscope using magnification 40×/1.15 NA oil.
3
Histopathological and Vascular Analysis of Tibialis Anterior Muscle
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For histopathological analysis, right and left TA muscles were collected and snap-frozen in liquid nitrogen-cooled isopentane. Six different levels of 7 μm-thick sections were cut and stained with hematoxylin-eosin (HE) to describe histological alterations, Von Kossa to evaluate calcium deposition, and Sirius red to visualise collagen deposits.
To preserve GFP fluorescence for the study of blood vessel 3D organisation, whole TA muscles were fixed in 10% neutral buffered formalin for 2 h, then cryopreserved in 40% sucrose overnight at 4°C before freezing in OCT (Tissue-Tek®, Sakura® Finetek, CA, USA) in small cryomolds. Serial cryosections (7 μm or 100 μm-thick sections for 2D and 3D analysis, respectively) were performed.
Images were captured on a Nikon Eclipse E800 microscope with Nikon ACT-1 software and DXM1200 camera for bright field. Fluorescence images and 3D reconstructions were performed with Leica® TCS SPE DM 2500 and LAS AF software (Leica®, Germany).
To preserve GFP fluorescence for the study of blood vessel 3D organisation, whole TA muscles were fixed in 10% neutral buffered formalin for 2 h, then cryopreserved in 40% sucrose overnight at 4°C before freezing in OCT (Tissue-Tek®, Sakura® Finetek, CA, USA) in small cryomolds. Serial cryosections (7 μm or 100 μm-thick sections for 2D and 3D analysis, respectively) were performed.
Images were captured on a Nikon Eclipse E800 microscope with Nikon ACT-1 software and DXM1200 camera for bright field. Fluorescence images and 3D reconstructions were performed with Leica® TCS SPE DM 2500 and LAS AF software (Leica®, Germany).
4
Visualizing Cell Spreading on Nanofibrous Membranes
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The spreading and morphology of the cells on uncoated or protein-coated nanofibrous membranes were visualized on days 1, 3, and 7 after seeding by staining the cells with a combination of fluorescent dyes diluted in PBS (5 μg/mL Hoechst 33258 cell nucleus dye; Sigma-Aldrich; and 20 ng/mL Texas red C2-maleimide cell membrane dye; Thermo Fisher Scientific) for 1 hour at room temperature in the dark. Instead of Texas red staining, the F-actin cytoskeleton of the cells was stained with phalloidin conjugated with tetramethylrhodamine isothiocyanate fluorescent dye (Sigma-Aldrich), diluted in PBS to a final concentration of 5 μg/mL, for 1 hour at room temperature in the dark. Before staining, the cells were rinsed with PBS and were fixed with −20°C cold ethanol for 10 minutes. Images of the cells were taken using epifluorescence microscopy (magnification 10×, IX 51; Olympus, Tokyo, Japan) equipped with a digital camera (DP 70), or using the Leica TCS SPE DM2500 upright confocal micro-scope, magnification 40×/1.15 NA oil. On day 1 after seeding of the cells, the spreading area of islands formed by human HaCaT keratinocytes was measured on images taken under fluorescence microscopy using the Atlas software.
5
Quantitative Analysis of Muscle Vasculature
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Images were captured on a Nikon Eclipse E800 microscope using the Nikon ACT-1 software and DXM1200 camera for bright field image acquisition. Fluorescence images and 3D reconstructions were performed with a Leica® TCS SPE DM 2500 and LAS AF software (Leica®, Germany).
Two-dimension analyses were performed, using ImageJ (NIH, MA, USA) and NIS-Element (Nikon) software. We measured the muscle fibers’ count, diameter, and capillary number per myofiber. At least 100 randomly selected fibers were considered for each muscle.
Three-dimensional analysis was performed to evaluate the organization of the vascular network and to quantify the number of vessels sprouting. For each muscle, 10 images were collected at 4 μm intervals to create a stack in the z axis. 3D reconstruction of this z-stack image was performed using 80- to 150-μm-thick frozen sections.
Two-dimension analyses were performed, using ImageJ (NIH, MA, USA) and NIS-Element (Nikon) software. We measured the muscle fibers’ count, diameter, and capillary number per myofiber. At least 100 randomly selected fibers were considered for each muscle.
Three-dimensional analysis was performed to evaluate the organization of the vascular network and to quantify the number of vessels sprouting. For each muscle, 10 images were collected at 4 μm intervals to create a stack in the z axis. 3D reconstruction of this z-stack image was performed using 80- to 150-μm-thick frozen sections.
6
Evaluation of Collagen Scaffold Morphology
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The morphology of both the unmineralized collagen scaffolds (UCS) and the mineralized collagen scaffolds (MCS) was evaluated by scanning confocal microscopy. Both the UCS and the MCS were measured in two states—dry and wet. The wet condition better mimics the cell cultivation environment, and the samples were therefore immersed in a physiological solution for 3 days. After that, the dry and wet samples were optically sectioned by an upright scanning confocal microscope (Leica TCS SPE DM2500; Leica, Wetzlar, Germany), obj. ACS APO 10x/0.30 CS and C PLAN 4x/0.10. Optical sections were acquired from the top surface structures to a depth of 490 µm with a step size of approx. 22 µm. The samples were excited at 405 nm, and the autofluorescence signal was detected at 450 to 600 nm. The size of the pore entrances (entering pore area) was measured manually using Fiji software on microphotographs taken in 8 randomly selected regions for each type of scaffold. In each picture, 15 randomly selected pores were measured (i.e., 120 pores in total for each type of scaffold).
In addition, the morphology of the scaffolds was evaluated by scanning electron microscopy (SEM). The samples of the scaffolds were fixed by a double-faced adhesive tape to the holders and were evaluated in a Phenom G2 Scanning Electron Microscope (Phenom-World BV, Eindhoven, The Netherlands).
In addition, the morphology of the scaffolds was evaluated by scanning electron microscopy (SEM). The samples of the scaffolds were fixed by a double-faced adhesive tape to the holders and were evaluated in a Phenom G2 Scanning Electron Microscope (Phenom-World BV, Eindhoven, The Netherlands).
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