Hemostasis, the process of normal physiological control of vascular damage, is fundamental to human life. We all suffer minor cuts and puncture wounds from time to time. In hemostasis, self-limiting platelet aggregation leads to the formation of a structured thrombus in which bleeding cessation comes from capping the hole from the outside. Detailed characterization of this structure could lead to distinctions between hemostasis and thrombosis, a case of excessive platelet aggregation leading to occlusive clotting. An imaging-based approach to puncture wound thrombus structure is presented here that draws upon the ability of thin-section electron microscopy to visualize the interior of hemostatic thrombi. The most basic step in any imaging-based experimental protocol is good sample preparation. The protocol provides detailed procedures for preparing puncture wounds and platelet-rich thrombi in mice for subsequent electron microscopy. A detailed procedure is given for in situ fixation of the forming puncture wound thrombus and its subsequent processing for staining and embedding for electron microscopy. Electron microscopy is presented as the end imaging technique because of its ability, when combined with sequential sectioning, to visualize the details of the thrombus interior at high resolution. As an imaging method, electron microscopy gives unbiased sampling and an experimental output that scales from nanometer to millimeters in 2 or 3 dimensions. Appropriate freeware electron microscopy software is cited that will support wide-area electron microscopy in which hundreds of frames can be blended to give nanometer-scale imaging of entire puncture wound thrombi cross-sections. Hence, any subregion of the image file can be placed easily into the context of the full cross-section.
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http://dx.doi.org/10.3791/66479 | DOI Listing |
Methods Mol Biol
January 2025
Cambridge Institute for Medical Research (CIMR) and Department of Clinical Biochemistry, University of Cambridge School of Clinical Medicine, Cambridge, UK.
Electron tomography can provide additional morphological information not easily obtained by conventional transmission electron microscopy of thin sections. It uses a goniometer stage in the electron microscope to tilt the specimen and collect a series of 2D images from different orientations, which are combined to provide a 3D volume tomogram and a colored reconstruction of the morphological feature(s) of interest. Here we describe the protocols for its use in visualizing changes in organelle morphology after depletion of the SNARE proteins VAMP7 and VAMP8 and to study VAMP7 localization on endolysosomes/lysosomes.
View Article and Find Full Text PDFMethods Mol Biol
January 2025
Department of Biochemistry, University of Toronto, Toronto, ON, Canada.
Negative staining electron microscopy is one of the easiest ways to determine the shape and dimensions of multimeric protein complexes over 100 kDa molecular weight. This method requires small volumes (< 10 μL) of dilute protein (0.01-0.
View Article and Find Full Text PDFAesthetic Plast Surg
January 2025
Department of Plastic and Reconstruction Surgery, Xijing Hospital, Fourth Military Medical University, Xi'an, Shaanxi, China.
Background: External volume expansion (EVE) devices has been demonstrated to enhance the survival of fat grafts. Decellularized adipose tissue (DAT) serves as a promising scaffold for adipose regeneration; however, the effectiveness of adipose regeneration in DAT remains limited, and the underlying mechanisms of its regeneration require further investigation.
Objective: This study explores the potential of EVE technology to enhance DAT-mediated adipogenesis by facilitating cellular recruitment and establishing a microenvironment conducive to adipose tissue regeneration.
Sci Rep
January 2025
State Key Laboratory of Oil and Gas Reservoir Geology and Exploitation, Southwest Petroleum University, Chengdu, 610500, China.
The exploration and development of deep marine shale gas has made significant breakthroughs, but factors influencing gas contents of deep marine shale are elusive, and quantitative prediction methods of gas content needs to be refined urgently. In this study, the deep marine shale of Longmaxi Formation in Luzhou area was taken as an example, vitrinite reflectance analysis, kerogen microscopy experiment, TOC content analysis, mineral composition analysis, gas content measurement, isothermal adsorption experiment, physical property analysis and argon ion polishing scanning electron microscopy experiment were carried out to find out factors affecting the gas content of deep marine shale, and a gas content prediction model has been worked out. Conclusions below have been reached: the content of adsorbed gas is mainly affected by Ro, TOC content, porosity, water saturation, clay mineral content, formation temperature and pressure; the content of free gas is mainly controlled by porosity, water saturation, formation temperature and pressure; according to the prediction models, the adsorbed gas content, free gas content and total gas content of each well were quantitatively calculated, and the study area was divided into Class I (with a total gas content ≥ 11 m/t), Class II (with a total gas content between 9 m/t and 11 m/t), and Class III (with a total gas content < 9 m/t) gas-bearing areas.
View Article and Find Full Text PDFNat Struct Mol Biol
January 2025
Key Laboratory of Biomacromolecules (CAS), National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.
The vesicular acetylcholine transporter (VAChT) has a pivotal role in packaging and transporting acetylcholine for exocytotic release, serving as a vital component of cholinergic neurotransmission. Dysregulation of its function can result in neurological disorders. It also serves as a target for developing radiotracers to quantify cholinergic neuron deficits in neurodegenerative conditions.
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