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Using C. elegans as a Model in PKD
Published in Jinghua Hu, Yong Yu, Polycystic Kidney Disease, 2019
ProtocolUse glow discharge formvar/carbon coated nickel grids (200 mesh, EMS cat no. FCF200-Ni). For negative staining; any type of formvar/carbon coated grids would work. Nickel grids are used here because gold or copper grids are not compatible with the following silver enhancement immunogold labeling protocol.Add 5 μL EV sample onto each grid, wait for 30 s, wick the solution with #1 Whitman filter paper from the edge of the grid, leaving a thin film of solution on the grid. Tilt the grid to get rid of remaining solution. However, do not touch the grid with filter paper, and do not let the grid dry before next step.Immediately add a drop Nano-W (Methylamine Tungstate solution nanoprobes cat no. 2018: http://www.nanoprobes.com/products/Negative-Stains.html) on the grid, wait for 1 min, wick the solution with Whitman filter paper, leave as little solution on grid as possible, but do not touch the grid with filter paper. Air dry and save the grid in a clean box for TEM.
Hormones of the Moss Protonema
Published in R. N. Chopra, Satish C. Bhatla, Bryophyte Development: Physiology and Biochemistry, 2019
In a very recent paper the distribution of IPA within the protonema cells of OVE mutants of Physcomitrella was studied using a monoclonal antibody with high affinity to IPA. After fixation and embedding of the material at low temperature the position was visualized by an indirect immunogold labeling. Only the cell wall of a part of all cells was labeled.94 Perhaps this detected only the cytokinin which left the cell and was, therefore, present for a short time in the wall. In the substrate cytokinins can always be found in high amounts.86,87,91
Collagens of the Disc
Published in Peter Ghosh, The Biology of the Intervertebral Disc, 2019
Presumably, types I and II collagen molecules are each restricted to their own homopolymeric fibrils. However, this has not been confirmed, for example, using type-specific antibodies and immunogold labeling under the electron microscope. Biochemical analyses designed to test for possible molecular intermingling, by seeking intertype cross-linked collagen peptides in protease digests of bovine annulus fibrosus, have so far failed to turn up any type I-type II hybrids.55 Nevertheless, it remains possible that hybrid fibrils, which are copolymers of types I and II collagen molecules, are present in the annulus fibrosus.
Metal Nanoparticles in Infection and Immunity
Published in Immunological Investigations, 2020
Review of the literature showed that many of the articles retrieved described using metal nanoparticles in vitro in diagnostic tests, including those for infectious diseases. This is not a surprise because magnetic beads have been used for many years in immunoassays, and other diagnostic assays, because of their ability to help separate a target molecule (such as a DNA sequence or a protein) from background material in the sample. Immunogold labeling of antibodies has been used for many years in electron microscopy. Figure 1b shows that there are at least four separate categories of articles on metal nanoparticles related to infection and immunity. This review will completely omit the articles using metal nanoparticles for in vitro diagnosis alone, and will only briefly mention articles which used nanoparticles as carriers for other known antibiotics or antimicrobials. (Figure 1.)
Use of electron microscopy to study platelets and thrombi
Published in Platelets, 2020
Maurizio Tomaiuolo, Rustem I. Litvinov, John W. Weisel, Timothy J. Stalker
Immunogold labeling is a staining technique that helps to localize cellular structures and macromolecules by loading antibodies or other specifically targeted proteins with colloidal gold particles of nanometer size. Gold is used for its high electron density that increases electron scatter to give dark spots in the TEM and SEM images. This technique has been applied successfully in many platelet studies. For example, immunogold labeling was used to demonstrate the spatial distribution of the GPIb-IX-V complex and αIIbβ3 on resting and activated platelets (reviewed in [41]). Immunogold labeling in combination with TEM has been used to determine the subcellular localization of many platelet proteins, such as matrix metalloproteinase-9, which was found on the plasma membrane, α-granules, open canalicular system, and within the cytoplasm both in resting and activated platelets [42]. Immunogold labeling of the purine P2Y1 receptor and the thromboxane-prostanoid TP receptor revealed that, while present at the platelet surface, both receptors were also abundantly represented inside the platelet [43]. Immunogold labeling has also been critical for examining platelet α-granule constituents, providing evidence of heterogeneous localization of various protein constituents, such as von Willebrand factor and fibrinogen, within individual α-granules [44,45].
Selective release of circRNAs in platelet-derived extracellular vesicles
Published in Journal of Extracellular Vesicles, 2018
Christian Preußer, Lee-Hsueh Hung, Tim Schneider, Silke Schreiner, Martin Hardt, Anna Moebus, Sentot Santoso, Albrecht Bindereif
Electron microscopy was performed on EV pellets stored at −80°C. For whole-mount analysis, EV suspensions (5 µL each) were incubated for 1 min on formvar and carbon-coated glow-discharged copper grids and subsequently stained with 2% uranyl acetate. Immunogold labelling was performed as described [22–24]. After blocking with 1% BSA in phosphate-buffered saline, grids were incubated with a 1:50 dilution of primary mouse anti-CD63 antibody (Thermo Fisher Scientific; TS63) in PBS containing 0.1% BSA for 1 h at room temperature. Extensive washes in the same buffer were followed by incubation with rabbit-anti-mouse antibody (Jackson ImmunoResearch) for 30 min, and after repeated washes 5-nm Protein A-gold (UMC Utrecht, the Netherlands) was applied for 1 h. After subsequent thorough washing, preparations were fixed using 1% glutaraldehyde in PBS, washed again and finally stained with 2% uranyl acetate. For controls, the primary antibody was omitted, resulting in complete loss of labelling (Supplementary figure S1). Preparations were inspected in an EM912 AB transmission electron microscope (Zeiss) at 120 kV under zero-loss conditions at slight underfocus. Images were recorded using a 2k x 2k slow-scan CCD camera (TRS, Germany) and the iTEM software package (Olympus-SIS).