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Acta Histochemica Et Cytochemica Feb 2018Combined analysis of immunostaining for various biological molecules coupled with investigations of ultrastructural features of individual cells is a powerful approach...
Combined analysis of immunostaining for various biological molecules coupled with investigations of ultrastructural features of individual cells is a powerful approach for studies of cellular functions in normal and pathological conditions. However, weak antigenicity of tissues fixed by conventional methods poses a problem for immunoassays. This study introduces a method of correlative light and electron microscopy imaging of the same endocrine cells of compact and diffuse islets from human pancreatic tissue specimens. The method utilizes serial sections obtained from Epon-embedded specimens fixed with glutaraldehyde and osmium tetroxide. Double-immunofluorescence staining of thick Epon sections for endocrine hormones (insulin and glucagon) and regenerating islet-derived gene 1 α (REG1α) was performed following the removal of Epoxy resin with sodium ethoxide, antigen retrieval by autoclaving, and de-osmification treatment with hydrogen peroxide. The immunofluorescence images of endocrine cells were superimposed with the electron microscopy images of the same cells obtained from serial ultrathin sections. Immunofluorescence images showed well-preserved secretory granules in endocrine cells, whereas electron microscopy observations demonstrated corresponding secretory granules and intracellular organelles in the same cells. In conclusion, the correlative imaging approach developed by us may be useful for examining ultrastructural features in combination with immunolocalisation of endocrine hormones in the same human pancreatic islets.
PubMed: 29622846
DOI: 10.1267/ahc.17020 -
Journal of Microscopy and Ultrastructure 2017Apoptosis is a tightly programmed cell suicide which occurs in multiple physiologic and pathological conditions where it plays an important role in tissue development...
Apoptosis is a tightly programmed cell suicide which occurs in multiple physiologic and pathological conditions where it plays an important role in tissue development and homeostasis by eliminating unwanted and damaged cells. Appropriate apoptosis signalling is crucial in maintaining the fine balance between cell death and cell survival in cancer. In response to death stimuli the morphology of the cell undergoes unique changes. The aim of this study was to examine and compare the changes in the cell surface morphology using scanning electron microscopy in HCS-2 cells, following 24 hour treatment with components of highly active antiretroviral therapy (HAART) at their clinical plasma concentrations. The cells were fixed in 2.5% Glutaraldehyde and post-fixed in 1% osmium tetroxide. The cells were then dehydrated through a graded series of alcohol and treated with hexamethyl-disilazane, then coated with a double layer of carbon. The cells were viewed under a Zeiss Ultra FEG Scanning Electron Microscope and a one way ANOVA and Tukey Kramer test was conducted based on the scoring of surface morphology of the cells using JMP 11 statistical software. The drugs used in this study induced morphological features which are known to be characteristic of apoptotic cell death. The drug combinations (ATP and LPV/r) were seemingly more effective than individual treatments in inducing cell death because morphological features observed were more advanced than those observed in individual treatments. However, LPV/r was more potent than ATP. In conclusion, HAART showed anticancer properties by inducing cell death through apoptosis.
PubMed: 30023236
DOI: 10.1016/j.jmau.2016.06.001 -
PloS One 2017Sample preparation is critical to biological electron microscopy (EM), and there have been continuous efforts on optimizing the procedures to best preserve structures of...
Sample preparation is critical to biological electron microscopy (EM), and there have been continuous efforts on optimizing the procedures to best preserve structures of interest in the sample. However, a quantitative characterization of the morphological changes associated with each step in EM sample preparation is currently lacking. Using correlative EM and superresolution microscopy (SRM), we have examined the effects of different drying methods as well as osmium tetroxide (OsO4) post-fixation on cell morphology during scanning electron microscopy (SEM) sample preparation. Here, SRM images of the sample acquired under hydrated conditions were used as a baseline for evaluating morphological changes as the sample went through SEM sample processing. We found that both chemical drying and critical point drying lead to a mild cellular boundary retraction of ~60 nm. Post-fixation by OsO4 causes at least 40 nm additional boundary retraction. We also found that coating coverslips with adhesion molecules such as fibronectin prior to cell plating helps reduce cell distortion from OsO4 post-fixation. These quantitative measurements offer useful information for identifying causes of cell distortions in SEM sample preparation and improving current procedures.
Topics: Animals; Cell Line; Cloning, Molecular; Microscopy, Electron, Scanning; Specimen Handling
PubMed: 28562683
DOI: 10.1371/journal.pone.0176839 -
Obesity (Silver Spring, Md.) Jan 2017To determine whether adipocyte diameters from three measurement methods are similarly associated with adiposity measurements and cardiometabolic variables. (Comparative Study)
Comparative Study
OBJECTIVE
To determine whether adipocyte diameters from three measurement methods are similarly associated with adiposity measurements and cardiometabolic variables.
METHODS
Surgical samples of omental and abdominal subcutaneous adipose tissue were obtained in a sample of 60 women (age 35-59 years; body mass index 20.3-41.1 kg/m ). Median adipocyte diameter of the main cell population was determined by collagenase digestion, osmium tetroxide fixation, and histological analysis. Adiposity and cardiometabolic risk factors were assessed.
RESULTS
Adipocyte diameter was consistently smaller with formalin fixation than with collagenase digestion, whereas osmium-fixed cells were larger (P < 0.0001, for all). Median adipocyte diameters derived from all methods were intercorrelated (r = 0.46-0.83, P < 0.001 for all). Positive associations were found between adipocyte diameters from all techniques and regional or total adiposity measurements (P < 0.01 for all). Omental adipocyte diameter was positively associated with fasting glucose, insulin, and homeostatic model assessment of insulin resistance (r = 0.30-0.52, P < 0.05 for all), with osmium-fixed cell size as a stronger correlate. Osmium-fixed cell diameter was also a better correlate of plasma adiponectin and leptin.
CONCLUSIONS
Although measurement techniques generated systematic differences in adipocyte size, associations with adiposity were only slightly affected by the technique. Osmium fixation generated stronger associations with cardiometabolic risk factors than collagenase digestion and histological analysis.
Topics: Adipocytes; Adipokines; Adiposity; Adult; Blood Glucose; Body Composition; Body Mass Index; Cardiovascular Diseases; Cell Size; Cholesterol; Female; Humans; Insulin; Insulin Resistance; Metabolic Syndrome; Middle Aged; Omentum; Risk Factors; Subcutaneous Fat, Abdominal; Triglycerides; Waist Circumference
PubMed: 27883275
DOI: 10.1002/oby.21697 -
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 -
The Science of the Total Environment May 2018Diet shifts are common in mammals and birds, but little is known about how such shifts along the food web affect contaminant exposure. Voles are staple food for many...
Diet shifts are common in mammals and birds, but little is known about how such shifts along the food web affect contaminant exposure. Voles are staple food for many mammalian and avian predators. There is therefore a risk of transfer of contaminants accumulated in voles within the food chain. Osmium is one of the rarest earth elements with osmium tetroxide (OsO) as the most toxic vapor-phase airborne contaminant. Anthropogenic OsO accumulates in fruticose lichens that are important winter food of bank voles (Myodes glareolus). Here, we test if a) anthropogenic osmium accumulates in bank voles in winter, and b) accumulation rates and concentrations are lower in autumn when the species is mainly herbivorous. Our study, performed in a boreal forest impacted by anthropogenic osmium, supported the hypotheses for all studied tissues (kidney, liver, lung, muscle and spleen) in 50 studied bank voles. In autumn, osmium concentrations in bank voles were even partly similar to those in the graminivorous field vole (Microtus agrestis; n=14). In autumn but not in late winter/early spring, osmium concentrations were generally negatively correlated with body weight and root length of the first mandible molar, i.e. proxies of bank vole age. Identified negative correlations between organ-to-body weight ratios and osmium concentrations in late winter/early spring indicate intoxication. Our results suggest unequal accumulation risk for predators feeding on different cohorts of bank voles.
Topics: Air Pollutants; Animals; Arvicolinae; Diet; Food Chain; Nutritional Status; Osmium; Seasons; Sweden
PubMed: 29079088
DOI: 10.1016/j.scitotenv.2017.10.056 -
BioTechniques Dec 2014The high-order structure of human chromosomes is an important biological question that is still under investigation. Studies have been done on imaging human mitotic...
The high-order structure of human chromosomes is an important biological question that is still under investigation. Studies have been done on imaging human mitotic chromosomes using mostly 2-D microscopy methods. To image micron-sized human chromosomes in 3-D, we developed a procedure for preparing samples for serial block-face scanning electron microscopy (SBFSEM). Polyamine chromosomes are first separated using a simple filtration method and then stained with heavy metal. We show that the DNA-specific platinum blue provides higher contrast than osmium tetroxide. A two-step procedure for embedding chromosomes in resin is then used to concentrate the chromosome samples. After stacking the SBFSEM images, a familiar X-shaped chromosome was observed in 3-D.
Topics: Cell Line; Chromosomes, Human; Coloring Agents; Histocytological Preparation Techniques; Humans; Male; Microscopy, Electron, Scanning; Mitosis; Platinum
PubMed: 25495730
DOI: 10.2144/000114236 -
Adipocyte Dec 2019Intramuscular fat (IMF) accumulates in muscles of the rotator cuff after tendon tear. The number and cross-sectional area of fat clumps and of adipocytes were quantified...
Intramuscular fat (IMF) accumulates in muscles of the rotator cuff after tendon tear. The number and cross-sectional area of fat clumps and of adipocytes were quantified on osmium tetroxide stained sections of the proximal, middle and distal quarters of SSP muscles 4, 8 and 12 weeks after SSP tendon division in a rabbit model. Linear mixed-effects models were fitted to the data and statistical significance was evaluated by ANOVA. Both the number (P<0.001) and cross-sectional area (P<0.0005) of fat clumps increased after tendon detachment while time had no significant effect (both at P>0.01). IMF accumulation was more important in the distal quarter of detached SSP muscle near tendon sectioning and characterized by increases of the number (P<0.0005) and cross-sectional area of fat clumps (P<0.0005) compared to the proximal quarter. Adipocyte number increased after tendon detachment (P<0.0005) and over time (P<0.01). The cross-sectional area of adipocytes increased in the detached group compared to controls (P<0.01) while time had no significant effect (P>0.01). Interestingly, the number of adipocytes in the distal quarter increased (P<0.0005) but the cross-sectional area was smaller (P<0.0005) compared to adipocytes in the proximal quarter. Adipocyte hyperplasia localized near tendon sectioning was the main contributor to fat accumulation in the detached SSP muscles.
Topics: Adipocytes; Animals; Female; Hyperplasia; Muscle, Skeletal; Rabbits; Rotator Cuff Injuries
PubMed: 31033395
DOI: 10.1080/21623945.2019.1609201 -
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 -
The Journal of Endocrinology Jun 2016Starvation induces low bone mass and high bone marrow adiposity in humans, but the underlying mechanisms are poorly understood. The adipokine leptin falls in starvation,...
Starvation induces low bone mass and high bone marrow adiposity in humans, but the underlying mechanisms are poorly understood. The adipokine leptin falls in starvation, suggesting that hypoleptinemia may be a link between negative energy balance, bone marrow fat accumulation, and impaired skeletal acquisition. In that case, treating mice with leptin during caloric restriction (CR) should reduce marrow adipose tissue (MAT) and improve bone mass. To test this hypothesis, female C57Bl/6J mice were fed a 30% CR or normal (N) diet from 5 to 10 weeks of age, with daily injections of vehicle (VEH), 1mg/kg leptin (LEP1), or 2mg/kg leptin (LEP2) (N=6-8/group). Outcomes included body mass, body fat percentage, and whole-body bone mineral density (BMD) via peripheral dual-energy X-ray absorptiometry, cortical and trabecular microarchitecture via microcomputed tomography (μCT), and MAT volume via μCT of osmium tetroxide-stained bones. Overall, CR mice had lower body mass, body fat percentage, BMD, and cortical bone area fraction, but more connected trabeculae, vs N mice (P<0.05 for all). Most significantly, although MAT was elevated in CR vs N overall, leptin treatment blunted MAT formation in CR mice by 50% vs VEH (P<0.05 for both leptin doses). CR LEP2 mice weighed less vs CR VEH mice at 9-10 weeks of age (P<0.05), but leptin treatment did not affect body fat percentage, BMD, or bone microarchitecture within either diet. These data demonstrate that once daily leptin bolus during CR inhibits bone marrow adipose expansion without affecting bone mass acquisition, suggesting that leptin has distinct effects on starvation-induced bone marrow fat formation and skeletal acquisition.
Topics: Adipose Tissue; Adiposity; Animals; Bone Density; Bone Marrow; Bone and Bones; Caloric Restriction; Energy Metabolism; Female; Humans; Leptin; Mice; Mice, Inbred C57BL; Recombinant Proteins; X-Ray Microtomography
PubMed: 27340200
DOI: 10.1530/JOE-15-0473