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Immunocytochemical Detection Systems
Published in Lars-Inge Larsson, Immunocytochemistry: Theory and Practice, 2020
Dextrans, glycogen, and polyvinylpyrrolidone (without heavy metals added) have themselves been employed as tracers (e.g., for capillary permeability) at the EM level.2,317 The diameters used in these studies varied from 4 to 30 nm. The polymers are not very electrondense per se, but they chelate lead and other metal stains, which enhances their detectability in TEM. Ainsworth recommends postfixation of tissue in osmic acid-potassium ferrocyanide and counterstaining of sections with bismuth subnitrate chelated by alkaline tartrate.2 Use of dextrans as (nonimmunological) tracers has been extended to FITC-dextrans.156,342 Kent and Wilson conjugated dextran 250 and dextran 70 to immunoglobulins (by a periodate method) and obtained immunocytochemical staining with the conjugates.169 Again, electron-density of the markers was obtained due to chelation of lead from the lead citrate stain. The dextran 250 particles used by Kent and Nilson varied from 25 to 40 nm in diameter, but occasional particles were as small as 10 nm in diameter.169 Dextran 70 particles were noted to be smaller, but also more irregular.169
ExperimentaL Oral Medicine
Published in Samuel Dreizen, Barnet M. Levy, Handbook of Experimental Stomatology, 2020
Samuel Dreizen, Barnet M. Levy
Proliferation of epithelium within apical granulomas appears to be a necessary precursor to cyst formation. Such proliferation has usually been attributed to a reaction to inflammation. Summers and Papadimitriou73 studied the ultrastructure of apical granulomas to establish whether the epithelial proliferation is related to a phagocytic function. Small sections of human periapical granulomas containing proliferating epithelium were incubated with a radiopaque marker (10% Thorotrast) and tissue culture medium (modified Hanks) at 37°C for 30 min. They were then fixed in 2.5% buffered glutaraldehyde, postfixed in buffered osmium tetroxide, and embedded in Araldite. Sections were stained with lead citrate and examined in an electron microscope. Ultrastructurally, the apical granulomas were comprised of squamous epithelium, connective tissue on which the epithelium rested, and inflammatory cells. After challenge with Thorotrast, vacuoles containing electron dense matter were seen in the proliferating epithelial cells. These cells appear to have the property of microphagocytosis that endows them with the capacity to eliminate cellular debris and to participate in cyst formation.
Identifying Nanotoxicity at the Cellular Level Using Electron Microscopy
Published in Suresh C. Pillai, Yvonne Lang, Toxicity of Nanomaterials, 2019
Kerry Thompson, Alanna Stanley, Emma McDermott, Alexander Black, Peter Dockery
To provide sufficient contrast to the sample, heavy metal salt staining is required. Unlike in light microscopy where different cellular components can be selectively stained, for electron microscopy stains are compounds or ions of high atomic number which scatter the electron beam and are only semi-selective. However, the interaction of such stains with certain chemical groups is known and should be considered depending on the nanoparticle material as they may not only enhance the contrast of the nanoparticles, but by attaching to them may increase their size. The standard TEM counterstains for biological samples are uranyl acetate followed by lead citrate. Uranyl acetate interacts with anionic compounds and binds strongly to phosphate groups, particularly those of nucleic acids and membrane phospholipids, in addition to sialic acid carboxyl groups of proteins and lipids. Lead citrate acts as a mordant for osmium tetroxide and uranyl acetate and therefore further enhances the contrast provided by them. It also binds to negatively charged amino acids of proteins, hydroxyl groups of carbohydrates, and phosphate groups of nucleic acids (Bozzola, 2014b).
ZO-2 favors Hippo signaling, and its re-expression in the steatotic liver by AMPK restores junctional sealing
Published in Tissue Barriers, 2022
Laura González-González, Helios Gallego-Gutiérrez, Dolores Martin-Tapia, José Everardo Avelino-Cruz, Christian Hernández-Guzmán, Sergio Israel Rangel-Guerrero, Luis Marat Alvarez-Salas, Erika Garay, Bibiana Chávez-Munguía, María Concepción Gutiérrez-Ruiz, Dinorah Hernández-Melchor, Esther López-Bayghen, Lorenza González-Mariscal
To determine the paracellular permeability of the liver to lanthanum, we followed a protocol previously described.38 Briefly, livers were first flushed with a physiological solution to clear the tissue from the blood. Then we employed a perfusion/fixation solution of 2.5% glutaraldehyde/4% lanthanum nitrate in 0.1 mol/L cacodylate buffer, pH 7.8. The lanthanum nitrate solution was prepared by dissolving lanthanum nitrate in water (8%, wt/vol) at 65°C and diluting 1:1 with 0.2 mol/L cacodylate buffer pH 7.8. After perfusion, livers were excised, cut into fragments, and incubated in the perfusion/fixation solution. Then, tissue samples were rinsed for 30 min in 0.1 mol/L cacodylates buffer/4% nitrate lanthanum and postfixed in 1% osmium tetroxide, containing 4% nitrate lanthanum, for 1 h at 4°C. Samples were then dehydrated in graded ethanol and propylene oxide, embedded in Polybed epoxy resins, and polymerized at 60°C for 24 h. Thin sections (60 nm) were stained at room temperature for 20 min with uranyl acetate and subsequently for 2 min with lead citrate before examination using a Jeol JEM-1011 transmission electron microscope.
Yishen capsule promotes podocyte autophagy through regulating SIRT1/NF-κB signaling pathway to improve diabetic nephropathy
Published in Renal Failure, 2021
Yuxiang Liu, Wenyuan Liu, Ziyuan Zhang, Yaling Hu, Xiaodong Zhang, Yanyan Sun, Qingqing Lei, Dalin Sun, Ting Liu, Yanjun Fan, Hui Li, Wujie Ding, Jingai Fang
Podocytes were incubated with different interventions in culture media for 24 h, and then the cells were washed with pre-cooled PBS and digested. The cells were collected and fixed in a mixture of 2.0% PFA and 2.5% glutaraldehyde overnight at 4 °C. After fixing, the cells were incubated in 1% aqueous osmium tetroxide and after five washes in cacodylate buffer (5 min each), the samples were dehydrated through a series of graded alcohols and embedded in Epon 812 medium. Semi-thin sections were cut on a microtome (Ultracut Reichert-Jung, Leica Microsystems, EM-UC7, Germany) and stained with uranyl citrate for 15 min. After washing, the samples were incubated with lead citrate for 7 min. Ultra-thin sections were cut with a diamond knife (Diatome, 80-HIS, Switzerland) and transferred to copper grids. Samples were then examined under a transmission electron microscope (Nippon Electronics).
Iron nanoparticle bio-interactions evaluated in Xenopus laevis embryos, a model for studying the safety of ingested nanoparticles
Published in Nanotoxicology, 2020
Patrizia Bonfanti, Anita Colombo, Melissa Saibene, Luisa Fiandra, Ilaria Armenia, Federica Gamberoni, Rosalba Gornati, Giovanni Bernardini, Paride Mantecca
For TEM analyses, embryos were randomly selected at the end of the FETAX assays and fixed in 2.0% paraformaldehyde and 0.2% glutaraldehyde in 0.1 M phosphate buffer at pH 7.4. After several washes in the same buffer, embryos were post-fixed in 1% OsO4 in 0.1 M phosphate buffer at pH 6.0 for 1.5 h at 4 °C in the dark, washed with milliQ water, counterstained with 1% uranyl acetate, dehydrated in a graded ethanol series, and transferred in 100% propylene oxide. Infiltration was performed with propylene oxide and embedding resin (Araldite-Epon) at volumetric proportions of 2:1 for 1.5 h, 1:1 overnight, 1:2 for 1.5 h and pure resin for 4 h. Samples were polymerized at 60 °C for 48 h. Ultra-thin sections were cut with a Reichert Ultracut E microtome and collected on 200-mesh uncoated copper grids. Sections were not counterstained to avoid contaminations by lead citrate and uranyl acetate that ultimately may interfere with metal NP visualization. Samples were analyzed using a Jeol JEM1220 transmission electron microscope operating at an accelerating voltage of 80 kV and equipped with a Lheritier LH72WA-TEM digital camera.