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Foods (Basel, Switzerland) Apr 2020The aim of the research was to verify the necessity of secondary fixation with osmium tetroxide in various types of meat products and evaluation of structural changes of...
The aim of the research was to verify the necessity of secondary fixation with osmium tetroxide in various types of meat products and evaluation of structural changes of products using different fixation procedures. The material for the study consisted of 11 types of meat products that were analyzed using a scanning electron microscope (SEM) with two different methods of chemical fixation. The first method included the usual processing of biological samples: glutaraldehyde primary fixation, the use of a buffer, secondary fixation by osmium tetroxide (OsO), buffer, and dehydration using ethanol of increasing concentrations. The second method comprised the glutaraldehyde primary fixation and dehydration using the ethanol of increasing concentrations only. The results unambiguously suggest that the main difference between these methods is in fixation and visibility of fat. Our analysis principally suggests that fixation of the product with OsO allows the tracking of all components (fat droplets, muscle fibers, connective tissue) in meat products. At the same time, our results also support the possibility that the secondary fixation can be skipped during the analysis, where the main objection is an observation of lipid-free structures of the meat products (e.g., connection between muscle and starches or spices) or meat products with an insignificant amount of fat.
PubMed: 32295008
DOI: 10.3390/foods9040487 -
Laboratory Investigation; a Journal of... Jan 2023Immunohistochemical analysis of formalin-fixed paraffin-embedded (FFPE) tissue blocks is routinely used to identify virus-infected cells. However, detecting virus...
Immunohistochemical analysis of formalin-fixed paraffin-embedded (FFPE) tissue blocks is routinely used to identify virus-infected cells. However, detecting virus particles in FFPE sections using light microscopy is difficult because of the light diffraction resolution limitations of an optical microscope. In this study, light microscopy and field emission scanning electron microscopy were performed to observe 3-dimensional virus particles in FFPE sections in a nondestructive manner using NanoSuit or osmium conductive treatment methods. The virus particles in FFPE sections were immunostained with specific antibodies against the surface antigens of the viral particles and stained with 3,3'-diaminobenzidine. A metal solution (0.2% gold chloride or 2% osmium tetroxide) was applied to enhance the 3,3'-diaminobenzidine-stained area. This procedure is nondestructive for FFPE sections and is a simpler method than transmission electron microscopy. To validate the applicability of this technique, we performed 3-dimensional imaging of the virus particles of different sizes, such as human papillomavirus, cytomegalovirus, and varicella-zoster virus. Furthermore, ultrathin sections from the FFPE sections that were observed to harbor viral particles using field emission scanning electron microscopy were prepared and assessed using transmission electron microscopy. In the correlative areas, transmission electron microscopy confirmed the presence of large numbers of virus particles. These results indicated that the combination of marking viral particles with 3,3'-diaminobenzidine/metal staining and conductive treatment can identify active progeny virus particles in FFPE sections using scanning electron microscopy. This easy correlative imaging of field emission scanning electron microscopy of the identical area of FFPE in light microscopy may help elucidate new pathological mechanisms of virus-related diseases.
Topics: Humans; Microscopy, Electron, Scanning; Paraffin Embedding; 3,3'-Diaminobenzidine; Formaldehyde; Virion
PubMed: 36748195
DOI: 10.1016/j.labinv.2022.100020 -
Scientific Reports Nov 2020Nanopores can serve as single molecule sensors. We exploited the MinION, a portable nanopore device from Oxford Nanopore Technologies, and repurposed it to detect any...
Nanopores can serve as single molecule sensors. We exploited the MinION, a portable nanopore device from Oxford Nanopore Technologies, and repurposed it to detect any DNA/RNA oligo (target) in a complex mixture by conducting voltage-driven ion-channel measurements. The detection and quantitation of the target is enabled by the use of a unique complementary probe. Using a validated labeling technology, probes are tagged with a bulky Osmium tag (Osmium tetroxide 2,2'-bipyridine), in a way that preserves strong hybridization between probe and target. Intact oligos traverse the MinION's nanopore relatively quickly compared to the device's acquisition rate, and exhibit count of events comparable to the baseline. Counts are reported by a publicly available software, OsBp_detect. Due to the presence of the bulky Osmium tag, probes traverse more slowly, produce multiple counts over the baseline, and are even detected at single digit attomole (amole) range. In the presence of the target the probe is "silenced". Silencing is attributed to a 1:1 double stranded (ds) complex that does not fit and cannot traverse this nanopore. This ready-to-use platform can be tailored as a diagnostic test to meet the requirements for point-of-care cell-free tumor DNA (ctDNA) and microRNA (miRNA) detection and quantitation in body fluids.
Topics: DNA; Diagnostic Tests, Routine; MicroRNAs; Nanopores; Nanotechnology; Osmium; Sequence Analysis, DNA
PubMed: 33188229
DOI: 10.1038/s41598-020-76667-1 -
Microscopy (Oxford, England) Apr 2022Precise immunolocalization of molecules in relation to ultrastructural features is challenging, especially when the target is small and not frequent enough to be...
Serial ultrathin sections to identify ultrastructural localization of GLUT1 molecules in vesicles in brain endothelial cells-correlative light and electron microscopy in depth.
Precise immunolocalization of molecules in relation to ultrastructural features is challenging, especially when the target is small and not frequent enough to be included in tiny ultrathin sections randomly selected for electron microscopy (EM). Glucose transporter 1 (GLUT1) is in charge of transporting glucose across brain capillary endothelial cells (BCECs). Paraformaldehyde-fixed floating sections (50 μm thick) of mouse brain were immunolabeled with anti-GLUT1 antibody and visualized with fluoronanogold. Fluorescent images encompassing the entire hemisphere were tiled to enable selection of GLUT1-positive BCECs suitable for subsequent EM and landmark placement with laser microdissection to guide trimming. Sections were then fixed with glutaraldehyde, gold enhanced to intensify the labeling and fixed with osmium tetroxide to facilitate ultrastructural recognition. Even though a region that contained target BCECs was successfully trimmed in the resin block, it was only after observation of serial ultrathin sections that GLUT1 signals in coated vesicles on the same cross section corresponding to the cross section preidentified by confocal laser microscope. This is the first ultrastructural demonstration of GLUT1 molecules in coated vesicles, which may well explain its functional relevance to transport glucose across BCECs. Successful ultrastructural localization of molecules in relation to well-preserved target structure in native tissue samples, as achieved in this study, will pave the way to understand the functional relevance of molecules and their relation to ultrastructural details.
Topics: Animals; Brain; Endothelial Cells; Glucose Transporter Type 1; Mice; Microscopy, Electron; Osmium Tetroxide
PubMed: 35157050
DOI: 10.1093/jmicro/dfac005 -
Scientific Reports Feb 2024Testing the hemocompatibility of medical devices after their interaction with blood entails the need to evaluate the activation of blood elements and the degree of their...
Testing the hemocompatibility of medical devices after their interaction with blood entails the need to evaluate the activation of blood elements and the degree of their coagulation and adhesion to the device surface. One possible way to achieve this is to use scanning electron microscopy (SEM). The aim was to develop a novel SEM-based method to assess the thrombogenic potential of medical devices and their adhesiveness to blood cells. As a part of this task, also find a convenient procedure of efficient and non-destructive sample fixation for SEM while reducing the use of highly toxic substances and shortening the fixation time. A polymeric surgical mesh was exposed to blood so that blood elements adhered to its surface. Such prepared samples were then chemically fixed for a subsequent SEM measurement; a number of fixation procedures were tested to find the optimal one. The fixation results were evaluated from SEM images, and the degree of blood elements' adhesion was determined from the images using ImageJ software. The best fixation was achieved with the May-Grünwald solution, which is less toxic than chemicals traditionally used. Moreover, manipulation with highly toxic osmium tetroxide can be avoided in the proposed procedure. A convenient methodology for SEM image analysis has been developed too, enabling to quantitatively evaluate the interaction of blood with the surfaces of various medical devices. Our method replaces the subjective assessment of surface coverage with a better-defined procedure, thus offering more precise and reliable results.
Topics: Microscopy, Electron, Scanning; Histological Techniques; Osmium Tetroxide
PubMed: 38409219
DOI: 10.1038/s41598-024-55136-z -
Biology Feb 2024Mice lacking () manifest reduced trabecular bone mass. However, the impact of expression in osteoblasts in vivo remains understudied. Herein, we generated...
Mice lacking () manifest reduced trabecular bone mass. However, the impact of expression in osteoblasts in vivo remains understudied. Herein, we generated osteoblast-specific transgenic (Tg) mice expressing and characterized their skeletal phenotype. Micro-CT analyses of the distal metaphysis of the femur showed a 50% and a 38% increase in trabecular bone mass in Tg male and female mice, respectively, due to a significant increase in trabecular number and a reduction in trabecular separation. Histomorphometry and serum biomarker studies uncovered that increased trabecular bone mass in Tg mice was the consequence of enhanced bone formation. Accordingly, an abundance of bone formation (, ), but not bone resorption (), markers were augmented in the femurs of Tg mice. Since the trabecular bone density is known to inversely correlate with the amount of marrow adipose tissue (MAT), we measured the MAT in osmium-tetroxide-labeled bones by micro-CT scanning. We found 86% less MAT in the proximal tibia of the Tg males. Consistently, the expression levels of the adipogenic markers, and , were 50% lower in the femurs of the Tg males. Our data are consistent with the possibility that claudin11 exerts anabolic effects in osteoblastic lineage cells that act via promoting the differentiation of marrow stem cells towards osteoblasts at the expense of adipocytes.
PubMed: 38392326
DOI: 10.3390/biology13020108 -
Neural Regeneration Research Sep 2019Recent studies have shown the potential of artificially synthesized conduits in the repair of peripheral nerve injury. Natural biopolymers have received much attention...
Recent studies have shown the potential of artificially synthesized conduits in the repair of peripheral nerve injury. Natural biopolymers have received much attention because of their biocompatibility. To investigate the effects of novel electrospun absorbable poly(ε-caprolactone)/type I collagen nanofiber conduits (biopolymer nanofiber conduits) on the repair of peripheral nerve injury, we bridged 10-mm-long sciatic nerve defects with electrospun absorbable biopolymer nanofiber conduits, poly(ε-caprolactone) or silicone conduits in Sprague-Dawley rats. Rat neurologica1 function was weekly evaluated using sciatic function index within 8 weeks after repair. Eight weeks after repair, sciatic nerve myelin sheaths and axon morphology were observed by osmium tetroxide staining, hematoxylin-eosin staining, and transmission electron microscopy. S-100 (Schwann cell marker) and CD4 (inflammatory marker) immunoreactivities in sciatic nerve were detected by immunohistochemistry. In rats subjected to repair with electrospun absorbable biopolymer nanofiber conduits, no serious inflammatory reactions were observed in rat hind limbs, the morphology of myelin sheaths in the injured sciatic nerve was close to normal. CD4 immunoreactivity was obviously weaker in rats subjected to repair with electrospun absorbable biopolymer nanofiber conduits than in those subjected to repair with poly(ε-caprolactone) or silicone. Rats subjected to repair with electrospun absorbable biopolymer nanofiber conduits tended to have greater sciatic nerve function recovery than those receiving poly(ε-caprolactone) or silicone repair. These results suggest that electrospun absorbable poly(ε-caprolactone)/type I collagen nanofiber conduits have the potential of repairing sciatic nerve defects and exhibit good biocompatibility. All experimental procedures were approved by Institutional Animal Care and Use Committee of Taichung Veteran General Hospital, Taiwan, China (La-1031218) on October 2, 2014.
PubMed: 31089062
DOI: 10.4103/1673-5374.255997 -
Heliyon Apr 2024Correlative light and electron microscopy (CLEM) combines light microscopy (LM) of fluorescent samples to ultrastructural analyses by electron microscopy (EM)....
Correlative light and electron microscopy (CLEM) combines light microscopy (LM) of fluorescent samples to ultrastructural analyses by electron microscopy (EM). Pre-embedding CLEM often suffers from inaccurate correlation between LM and EM modalities. Post-embedding CLEM enables precise registration of structures directly on EM sections, but requires fluorescent markers withstanding EM sample preparation, especially osmium tetroxide fixation, dehydration and EPON embedding. Most fluorescent proteins (FPs) lose their fluorescence during such conventional embedding (CE), but synthetic dyes represent promising alternatives as their stability exceeds those of FP. We analyzed various Janelia Fluor dyes and TMR conjugated to ligands for self-labeling enzymes, such as HaloTag, for fluorescence preservation after CE. We show that TMR, JF525, JF549, JFX549 and JFX554 retain fluorescence, with JFX549 and JFX554 yielding best results overall, also allowing integration of high-pressure freezing and freeze substitution. Furthermore, we found the recently published FP StayGold to resist CE, facilitating dual-fluorescence in-resin CLEM.
PubMed: 38560224
DOI: 10.1016/j.heliyon.2024.e28055 -
Molecules (Basel, Switzerland) Jun 2020Lignans are bioactive compounds that are especially abundant in the Norway spruce ( L. Karst.) knotwood. By combining a variety of chromatographic, spectroscopic and...
Lignans are bioactive compounds that are especially abundant in the Norway spruce ( L. Karst.) knotwood. By combining a variety of chromatographic, spectroscopic and imaging techniques, we were able to quantify, qualify and localise the easily extractable lignans in the xylem tissue. The knotwood samples contained 15 different lignans according to the gas chromatography-mass spectrometry analysis. They comprised 16% of the knotwood dry weight and 82% of the acetone extract. The main lignans were found to be hydroxymatairesinols HMR1 and HMR2. Cryosectioned and resin-embedded ultrathin sections of the knotwood were analysed with scanning transmission X-ray microscopy (STXM). Cryosectioning was found to retain only lignan residues inside the cell lumina. In the resin-embedded samples, lignan was interpreted to be unevenly distributed inside the cell lumina, and partially confined in deposits which were either readily present in the lumina or formed when OsO used in staining reacted with the lignans. Furthermore, the multi-technique characterisation enabled us to obtain information on the chemical composition of the structural components of knotwood. A simple spectral analysis of the STXM data gave consistent results with the gas chromatographic methods about the relative amounts of cell wall components (lignin and polysaccharides). The STXM analysis also indicated that a torus of a bordered pit contained aromatic compounds, possibly lignin.
Topics: Lignans; Microscopy, Electron, Scanning Transmission; Picea; Spectrometry, X-Ray Emission; X-Ray Microtomography
PubMed: 32630014
DOI: 10.3390/molecules25132997 -
Frontiers in Endocrinology 2024Unlike white adipose tissue depots, bone marrow adipose tissue (BMAT) expands during caloric restriction (CR). Although mechanisms for BMAT expansion remain unclear,...
Deficiency of glucocorticoid receptor in bone marrow adipocytes has mild effects on bone and hematopoiesis but does not influence expansion of marrow adiposity with caloric restriction.
INTRODUCTION
Unlike white adipose tissue depots, bone marrow adipose tissue (BMAT) expands during caloric restriction (CR). Although mechanisms for BMAT expansion remain unclear, prior research suggested an intermediary role for increased circulating glucocorticoids.
METHODS
In this study, we utilized a recently described mouse model () to exclusively target bone marrow adipocytes (BMAds) for elimination of the glucocorticoid receptor (GR) (i.e. ) whilst maintaining GR expression in other adipose depots.
RESULTS
Mice lacking GR in BMAds ( ) and control mice ( ) were fed or placed on a 30% CR diet for six weeks. On a normal chow diet, tibiae of female mice had slightly elevated proximal trabecular metaphyseal bone volume fraction and thickness. Both control and mice had increased circulating glucocorticoids and elevated numbers of BMAds in the proximal tibia following CR. However, no significant differences in trabecular and cortical bone were observed, and quantification with osmium tetroxide and μCT revealed no difference in BMAT accumulation between control or mice. Differences in BMAd size were not observed between and control mice. Interestingly, mice had decreased circulating white blood cell counts 4 h into the light cycle.
DISCUSSION
In conclusion, our data suggest that eliminating GR from BMAd has minor effects on bone and hematopoiesis, and does not impair BMAT accumulation during CR.
Topics: Animals; Receptors, Glucocorticoid; Caloric Restriction; Mice; Adipocytes; Adiposity; Female; Hematopoiesis; Bone Marrow; Mice, Knockout; Bone and Bones; Mice, Inbred C57BL; Adipose Tissue; Male; Metabolism, Inborn Errors
PubMed: 38887268
DOI: 10.3389/fendo.2024.1397081