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MethodsX 2022Toxicity evaluations involve the analysis of multiple biomarkers. In this study, the liver, target organ analyzed by treatments with iron concentrations, indicated the...
Toxicity evaluations involve the analysis of multiple biomarkers. In this study, the liver, target organ analyzed by treatments with iron concentrations, indicated the accumulation of lipids as a response. Considering that the distribution of lipids in an organ is directly related to the induction of inflammatory processes by aquatic contaminants, this study proposes to carry out an integrative investigation of the behavior and the distribution of lipids in the liver tissue. Techniques of light and electron microscopy were performed in order to propose a new way of assessing and quantifying the distribution of lipid droplets, also presenting methodological alternatives that can be chosen by the reader according to the interests and resources available. Thus, it is assumed that the method begins with the fixation of the liver with Glutaraldehyde 2,5% in PBS 0,1 M and continues with post fixation with osmium tretoxide 1%, which marks lipids. For this proposition, two inclusion methodologies were performed to histological analyses in Historesin and ultrastructural analyses in EMBeed 812. For light microscopy (LM) analyses, cuts were obtained with 2,5 micrometers thickness, which were stained with (1) Mayers hematoxylin and (2) toluidine blue. The images obtained were processed in software Image J Fiji to evidence the lipid distribution in liver.•Cytological reactions with osmium tetroxide constitute low complexity methods that allow the optimization of the localization, identification and quantification of lipid droplets in the liver tissue when analyzed under the conventional light microscope.•Samples included in EMBeed 812 resin commonly used in Transmission Electron Microscopy can be analyzed by SEM-BEC, as complementary analyses for the detection of lipids.•Using SEM-BEC and conventional light microscopy, it is possible to quantify the area occupied by lipid droplets using Image J Fiji software, as these are contrasted due to the reaction with osmium tetroxide.
PubMed: 35818446
DOI: 10.1016/j.mex.2022.101769 -
Beilstein Journal of Nanotechnology 2016Osmium tetroxide 2,2'-bipyridine (OsBp) is known to react with pyrimidines in ssDNA and preferentially label deoxythymine (T) over deoxycytosine (C). The product,...
Osmium tetroxide 2,2'-bipyridine (OsBp) is known to react with pyrimidines in ssDNA and preferentially label deoxythymine (T) over deoxycytosine (C). The product, osmylated DNA, was proposed as a surrogate for nanopore-based DNA sequencing due to OsBp's "perfect" label attributes. Osmylated deoxyoligos translocate unassisted and measurably slow via sub-2 nm SiN solid-state nanopores, as well as via the alpha-hemolysin (α-HL) pore. Both nanopores discriminate clearly between osmylated and intact nucleobase; α-HL was also shown to discriminate between osmylated T and osmylated C. Experiments presented here confirm that the kinetics of osmylation are comparable for short oligos and long ssDNA and show that pyrimidine osmylation is practically complete in two hours at room temperature with less than 15 mM OsBp. Under the proposed labeling conditions: deoxyoligo backbone degradation measures less than 1/1,000,000; false positives such as osmylated deoxyadenine (A) and osmylated deoxyguanine (G) measure less than 1/100,000; false negatives, i.e., unosmylated C measure less than 1/10,000; and unosmylated T must measure substantially lower than 1/10,000 due to the 27-fold higher reactivity of T compared to C. However, osmylated C undergoes degradation that amounts to about 1-2% for the duration of the labeling protocol. This degradation may be further characterized, possibly suppressed, and the properties of the degradation products via nanopore translocation can be evaluated to assure base calling quality in a DNA sequencing effort.
PubMed: 27826518
DOI: 10.3762/bjnano.7.135 -
Visualization of cytoplasmic organelles via in-resin CLEM using an osmium-resistant far-red protein.Scientific Reports Jul 2020Post-fixation with osmium tetroxide staining and the embedding of Epon are robust and essential treatments that are used to preserve and visualize intracellular...
Post-fixation with osmium tetroxide staining and the embedding of Epon are robust and essential treatments that are used to preserve and visualize intracellular membranous structures during electron microscopic analyses. These treatments, however, can significantly diminish the fluorescent intensity of most fluorescent proteins in cells, which creates an obstacle for the in-resin correlative light-electron microscopy (CLEM) of Epon-embedded cells. In this study, we used a far-red fluorescent protein that retains fluorescence after osmium staining and Epon embedding to perform an in-resin CLEM of Epon-embedded samples. The fluorescence of this protein was detected in 100 nm thin sections of the cells in Epon-embedded samples after fixation with 2.5% glutaraldehyde and post-fixation with 1% osmium tetroxide. We performed in-resin CLEM of the mitochondria in Epon-embedded cells using a mitochondria-localized fluorescent protein. Using this protein, we achieved in-resin CLEM of the Golgi apparatus and the endoplasmic reticulum in thin sections of the cells in Epon-embedded samples. To our knowledge, this is the first reported use of a far-red fluorescent protein retains its fluorescence after osmium staining and Epon-embedding, and it represents the first achievement of in-resin CLEM of both the Golgi apparatus and the endoplasmic reticulum in Epon-embedded samples.
Topics: Animals; COS Cells; Chlorocebus aethiops; Endoplasmic Reticulum; Fluorescence; Fluorescent Dyes; Golgi Apparatus; HEK293 Cells; HeLa Cells; Humans; Luminescent Proteins; Mitochondria; Osmium Tetroxide; Staining and Labeling; Red Fluorescent Protein
PubMed: 32647231
DOI: 10.1038/s41598-020-68191-z -
Protoplasma Nov 2020Bird feather lipids are usually attributed to the oily secretion product of the uropygial (preen) gland. We have observed, however, that feathers exhibit a strong...
Bird feather lipids are usually attributed to the oily secretion product of the uropygial (preen) gland. We have observed, however, that feathers exhibit a strong reaction with osmium tetroxide (OsO), even after treatment with detergents. This leads us to postulate the existence of endogenous feather lipids distinct from preen gland lipids. In order to substantiate our hypothesis, we investigated down feathers from a 1-day-old chicken as their uropgygial gland is not functionally active. The results confirmed the osmiophilic reaction, which was concentrated in the center of barbs and strongly reduced after lipid extraction. In these lipid extracts, we identified using thin layer chromatography, cholesterol, various ceramides, glycolipids, phospholipids, and fatty acids, which closely resembled the lipid composition of the water barrier in the chicken-cornified epidermal envelope. This composition is clearly distinct from chicken uropygeal gland secretion (UGS) known to consist of fatty alcohols as part of aliphatic monoester waxes and of free, predominantly saturated, fatty acids. A filter assay showed a strong reactivity between OsO and the fatty acids C18:1 and C18:2 and with feather lipid extracts, but not with UGS. These observations were confirmed by gas chromatography detecting unsaturated fatty acids including C18:1 and C18:2 as well as cholesterol exclusively in chicken feathers. Our results indicate that (1) endogenous lipids are detectable in chicken feathers and distinct from UGS and (2) in analogy to the morphogenesis of the cornified envelope of chicken feather lipids that may have derived from cellular feather-precursors, apparently enduring the specific cell death during developmental feather cornification.
Topics: Animals; Chickens; Feathers; Lipids; Sebaceous Glands
PubMed: 32851422
DOI: 10.1007/s00709-020-01544-7 -
Brazilian Journal of Otorhinolaryngology 2020The use of electron microscopy in the study of the inner ear has allowed us to observe minute details of the hair cells, especially in ototoxicity studies; however, the...
INTRODUCTION
The use of electron microscopy in the study of the inner ear has allowed us to observe minute details of the hair cells, especially in ototoxicity studies; however, the preparation of this material is a difficult and delicate task. In an attempt to simplify the handling of these materials, two agents, toluidine blue and ethylenediamine tetra-acetic acid were tested, in addition to the elimination of osmium tetroxide during the preparation of albino guinea pig cochleae. We also tested the applicability of these methodologies in an ototoxicity protocol.
OBJECTIVE
To verify the quality of the images obtained with and without the use of ethylenediamine tetra-acetic acid, toluidine blue and osmium tetroxide in the preparation of cochleae of albino guinea pigs for the scanning electron microscopy.
METHODS
Three groups of cochleae were used. In Group 1, 10 cochleae were prepared with the usual methodology, dissecting the optical capsule without decalcification and using osmium tetroxide as a post-fixative agent. In Group 2, we prepared 10 cochleae decalcified with ethylenediamine tetra-acetic acid, injecting toluidine blue in the endolymphatic space to facilitate the identification of the organ of Corti. In Group 3, we used 4 cochleae of guinea pigs that received 3 doses of cisplatin (7.5mg/kg, D1-D5-D6), two prepared according to the methodology used in Group 1 and two with that used in Group 2. Scanning electron microscopy images were obtained from the organ of Corti region of the basal turn of each cochlea.
RESULTS
The organ of Corti was more easily identified with the use of toluidine blue. The dissection of the cochlea was more accurate in the decalcified cochleae. The quality of the images and the preservation of the organ of Corti obtained with the two methodologies were similar.
CONCLUSION
The proposed modifications resulted in images of similar quality as those observed using the traditional methodology.
Topics: Animals; Cisplatin; Cochlea; Edetic Acid; Female; Guinea Pigs; Hair Cells, Auditory; Microscopy, Electron, Scanning; Organ of Corti; Osmium Tetroxide; Tolonium Chloride
PubMed: 30797727
DOI: 10.1016/j.bjorl.2018.11.008 -
Scientific Reports Jul 2022Biotin ligases have been developed as proximity biotinylation enzymes for analyses of the interactome. However, there has been no report on the application of proximity...
Biotin ligases have been developed as proximity biotinylation enzymes for analyses of the interactome. However, there has been no report on the application of proximity labeling for in-resin correlative light-electron microscopy of Epon-embedded cells. In this study, we established a proximity-labeled in-resin CLEM of Epon-embedded cells using miniTurbo, a biotin ligase. Biotinylation by miniTurbo was observed in cells within 10 min following the addition of biotin to the medium. Using fluorophore-conjugated streptavidin, intracellular biotinylated proteins were labeled after fixation of cells with a mixture of paraformaldehyde and glutaraldehyde. Fluorescence of these proteins was resistant to osmium tetroxide staining and was detected in 100-nm ultrathin sections of Epon-embedded cells. Ultrastructures of organelles were preserved well in the same sections. Fluorescence in sections was about 14-fold brighter than that in the sections of Epon-embedded cells expressing mCherry2 and was detectable for 14 days. When mitochondria-localized miniTurbo was expressed in the cells, mitochondria-like fluorescent signals were detected in the sections, and ultrastructures of mitochondria were observed as fluorescence-positive structures in the same sections by scanning electron microscopy. Proximity labeling using miniTurbo led to more stable and brighter fluorescent signals in the ultrathin sections of Epon-embedded cells, resulting in better performance of in-resin CLEM.
Topics: Biotin; Microscopy, Electron, Scanning; Organelles; Osmium Tetroxide; Resins, Plant; Staining and Labeling
PubMed: 35778550
DOI: 10.1038/s41598-022-15438-6 -
Sovremennye Tekhnologii V Meditsine 2021was to evaluate the efficacy of a novel technique for preparation, staining, and visualization of tissues containing extra-skeletal mineralization areas, all-metal...
UNLABELLED
was to evaluate the efficacy of a novel technique for preparation, staining, and visualization of tissues containing extra-skeletal mineralization areas, all-metal implants or their prototypes for their subsequent examination using scanning electron microscopy in the backscattered electron mode.
MATERIALS AND METHODS
After fixation in 10% formalin (24 h), the biomaterial (a titanium nickelide plate with the surrounding tissues after subcutaneous implantation, patented titanium alloy plates with the surrounding tissues after cranioplasty, primary and secondary calcified atherosclerotic plaques) were fixed with 1% osmium tetroxide (12 h) and then stained with 2% aqueous solution of osmium tetroxide (48 h). The samples were further stained with 2% alcoholic uranyl acetate (5 h), dehydrated with isopropanol (5 h) and acetone (1 h), impregnated with a mixture of acetone and epoxy resin Epon (1:1, 6 h) and then embedded into a fresh portion of epoxy resin (24 h), which was followed by polymerization at 60°C. After grinding and polishing, epoxy blocks were counterstained with lead citrate (7 min) and sputter-coated with carbon, then the samples were visualized by scanning electron microscopy in the backscattered electron mode. The elemental composition was studied using X-ray microanalysis.
RESULTS
The developed technique allows obtaining high-quality images at five thousand-fold magnifications, provides the possibility to identify the shape and structure of intact metal and mineral inclusions, and to type the surrounding cells, distinguishing mesenchymal and immunocompetent cells by shape and cytoplasmic content. Apart from connective tissue capsule thickness and leukocyte infiltration, this technique makes it possible to estimate the number and area of newly formed small-caliber vessels representing a surrogate marker of inflammation.
CONCLUSION
The proposed technique provides the possibility to investigate adequately the structure of samples when their sectioning is impossible or significantly complicated, with image quality remarkably higher than that obtained by light microscopy.
Topics: Alloys; Metals; Microscopy, Electron, Scanning; Osmium Tetroxide; Staining and Labeling
PubMed: 34795988
DOI: 10.17691/stm2020.12.4.02 -
Scientific Reports May 2020Fat embolism is the mechanical blockage of blood vessels by circulating fat particles. It is frequently related to traumas involving soft tissues and fat-containing... (Comparative Study)
Comparative Study
Fat embolism is the mechanical blockage of blood vessels by circulating fat particles. It is frequently related to traumas involving soft tissues and fat-containing bones. Different techniques have been used for decades to demonstrate histologically fat emboli, being the extremely toxic post-fixation with osmium tetroxide one of the most used techniques in the last decades. In the present study, the osmium tetroxide technique was compared qualitatively and quantitatively, for the first time, with chromic acid and Oil Red O frozen techniques for histological fat emboli detection in the lungs of eight sperm whales that died due to ship strikes. This was also the first time that chromic acid technique was tested in cetaceans. Results showed that the three techniques were valuable for the histological detection of fat embolism in cetaceans, even when tissues presented advanced autolysis and had been stored in formaldehyde for years. Although quantitative differences could not be established, the Oil Red O frozen technique showed the lowest quality for fat emboli staining. On the contrary, the chromic acid technique was proven to be a good alternative to osmium tetroxide due to its slightly lower toxicity, its equivalent or even superior capacity of fat emboli detection, and its significantly lower economic cost.
Topics: Animals; Cetacea; Embolism, Fat; Histological Techniques; Lung; Pulmonary Embolism; Staining and Labeling
PubMed: 32427895
DOI: 10.1038/s41598-020-64821-8 -
The Journal of Cell Biology Jan 1965A basis for the interpretation of the structure of the cell membrane is often looked for in electron microscope investigations on the structure of lipid models. A...
A basis for the interpretation of the structure of the cell membrane is often looked for in electron microscope investigations on the structure of lipid models. A difficulty in these investigations is our lack of knowledge of the effect of the preparative treatment on the structure studied. This applies especially to the strongly oxidizing fixatives: osmium tetroxide and potassium permanganate. These agents have, moreover, the drawback that they cannot be used to fix fully saturated lipids. On the basis of existing theories concerning complex colloid systems, a fixation method was developed that allows the electron microscope study of the structure of phospholipid models, irrespective of whether they consist of saturated or unsaturated compounds. With this fixation, namely tricomplex flocculation by means of suitable ions, the structure of the lipid molecules is not altered. Moreover, the site and mode of action of this fixation are well known. The first application of this method to the study of isolated beef brain phospholipids is described.
Topics: Biophysical Phenomena; Biophysics; Brain Chemistry; Cell Membrane; Cell Physiological Phenomena; Colloids; Electrons; Ferrocyanides; Histological Techniques; Ions; Lead; Lipids; Microscopy; Microscopy, Electron; Molybdenum; Nitrates; Osmium; Phospholipids; Research
PubMed: 14286294
DOI: 10.1083/jcb.24.1.23 -
Heliyon Jun 2023In-resin CLEM (Correlative Light and Electron Microscopy) of Epon-embedded cells involves correlating fluorescence microscopy with electron microscopy in the same...
In-resin CLEM (Correlative Light and Electron Microscopy) of Epon-embedded cells involves correlating fluorescence microscopy with electron microscopy in the same Epon-embedded ultrathin section. This method offers the advantage of high positional accuracy compared to standard CLEM. However, it requires the expression of recombinant proteins. In order to detect the localization of endogenous target(s) and their localized ultrastructures of Epon-embedded samples using in-resin CLEM, we investigated whether immunological and affinity-labeling using fluorescent dyes applied to in-resin CLEM of Epon-embedded cells. The orange fluorescent (λ ∼550 nm) and far-red (λ ∼650 nm) fluorescent dyes examined maintained a sufficient level of fluorescent intensity after staining with osmium tetroxide and subsequent dehydration treatment with ethanol. Immunological in-resin CLEM of mitochondria and the Golgi apparatus was achieved using anti-TOM20, anti-GM130 antibodies, and fluorescent dyes. Two-color in-resin CLEM revealed that wheat germ agglutinin-puncta showed the ultrastructures of multivesicular body-like structures. Finally, taking the advantage of high positional accuracy, volume in-resin CLEM of mitochondria in the semi-thin section (2 μm thick) of Epon-embedded cells was performed by focused ion beam scanning electron microscopy. These results suggested that the application of immunological reaction and affinity-labeling with fluorescent dyes to in-resin CLEM of Epon-embedded cells is suitable for analyzing the localization of endogenous targets and their ultrastructures by scanning and transmission electron microscopy.
PubMed: 37389060
DOI: 10.1016/j.heliyon.2023.e17394