Lead citrate

Manufactured by Electron Microscopy Sciences

Sourced in United States, Germany, United Kingdom, Czechia, Japan

Lead citrate is a chemical compound used as a staining agent in electron microscopy. It is primarily employed to enhance the contrast of biological samples during the imaging process. Lead citrate binds to specific cellular components, making them more visible under the electron microscope.

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Uranyl acetate and lead citrate (7 citations) Reynold’s lead citrate (6 citations) UranyLess and Lead Citrate (4 citations) Reynolds’s lead citrate (2 citations) Lead citrate solution (2 citations) Uranyl-acetate replacement stain and lead citrate (2 citations) Lead Citrate 3% (2 citations)

69 protocols using lead citrate

Mitochondrial Morphometric Analysis in Myocardium

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Mitochondrial morphology in myocardial tissues was assessed using transmission electron microscopy (TEM). The tissues were initially fixed in a 3% glutaraldehyde solution and subsequently post-fixed in 1% osmium tetroxide (Sigma-Aldrich). The samples were then dehydrated in an ascending acetone series and embedded in Epon 812 (SPI Supplies, West Chester, PA, USA). Ultrathin sections, with thicknesses ranging from 60–90 nm, were sliced with a diamond knife and stained with uranyl acetate (SPI Supplies) and lead citrate (Electron Microscopy Sciences, Hatfield, PA, USA). Images of interfibrillar mitochondria were captured at a magnification of 10,000 × using a JEM-1400-FLASH Transmission Electron Microscope (JOEL, Tokyo, Japan). An investigator, blinded to the experimental conditions, quantified various mitochondrial morphological parameters, such as area, perimeter, and circularity, using the ImageJ software [31 (link)].

Niu X., Zhang J., Hu S., Dang W., Wang K, & Bai M. (2024). lncRNA Oip5-as1 inhibits excessive mitochondrial fission in myocardial ischemia/reperfusion injury by modulating DRP1 phosphorylation. Cellular & Molecular Biology Letters, 29, 72.

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Transmission Electron Microscopy Sample Preparation

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Cells were fixed in 1% glutaraldeyde in sodium cacodylate buffer (0.2 M, pH 7.2) (Electron Microscopy Science), post-fixed in 1% buffered osmium tetroxide (Electron Microscopy Science), dehydrated in acetone (Merck), embedded in epoxy resin (Electron Microscopy Science) and polymerized at 60°C over the course of three days [25 , 26 , 27 , 28 (link)]. Ultrathin sections (50–70 nm thick) were obtained from the resin blocks. The sections were picked up using copper grids, stained with uranyl acetate (Electron Microscopy Science) and lead citrate (Electron Microscopy Science), and observed using a Jeol JEM 1011 transmission electron microscope.

Barreto-Vieira D.F., Jácome F.C., da Silva M.A., Caldas G.C., de Filippis A.M., de Sequeira P.C., de Souza E.M., Andrade A.A., Manso P.P., Trindade G.F., Lima S.M, & Barth O.M. (2017). Structural investigation of C6/36 and Vero cell cultures infected with a Brazilian Zika virus. PLoS ONE, 12(9), e0184397.

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Triglyceride and Glycogen Assay Protocol

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The Triglyceride Kit (GPO‐PAP Method) was purchased from BioSino Bio‐Technology & Science Incorporated (Beijing, China). The EnzyChrom™ Glycogen Assay Kit was obtained from BioAssay Systems (Hayward, CA, USA). Twenty‐five percent glutaraldehyde solution, 8% paraformaldehyde solution, EMbed 812 kit, uranyl acetate and lead citrate were all obtained from Electron Microscopy Sciences (Hatfield, PA, USA). Osmium tetraoxide (EM grade) was obtained from Nakalai Tesque (Kytoto, Japan). Potassium ferrocyanide was obtained from Sigma‐Aldrich (St Louis, MO, USA). Pierce™ BCA Protein Assay Kit, HCS LipidTOX™ Red Neutral Lipid Stain and MitoTracker™ Red CMXRos were obtained from Thermo Fisher Scientific (Waltham, MA, USA).

Liu Q., Zhou Z., Liu P, & Zhang S. (2019). Comparative proteomic study of liver lipid droplets and mitochondria in mice housed at different temperatures. Febs Letters, 593(16), 2118-2138.

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Cerebellar Ultrastructure Analysis

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Cerebella from perfused animals were postfixed in 2% glutaraldehyde (Sigma), followed by treatment with 1% osmium tetroxide (Sigma) and dehydration in ethanol and propylene oxide. Samples were then embedded in Epon (Fluka) and 70 nm sections were cut using an ultramicrotome (EM UC7, Leica). The sections were stained with uranyl acetate (PlanoGMBH) and lead citrate (Electron Microscopy Sciences). Images were taken by a transmission electron microscope (JEOL JEM2100PLUS) equipped with GATAN OneView camera.

Murru S., Hess S., Barth E., Almajan E.R., Schatton D., Hermans S., Brodesser S., Langer T., Kloppenburg P, & Rugarli E.I. (2019). Astrocyte‐specific deletion of the mitochondrial m‐AAA protease reveals glial contribution to neurodegeneration. Glia, 67(8), 1526-1541.

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Platelet Ultrastructure Analysis by TEM

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To analyse platelet ultrastructure, washed platelets were fixed with 2.5% glutaraldehyde (16,210, Electron Microscopy Sciences) in 50 mM cacodylate buffer (12,201, pH 7.2; AppliChem). After embedding in epon 812 (14,900, Electron Microscopy Sciences), ultrathin sections were generated and stained with 2% uranyl acetate (22,400, Electron Microscopy Sciences) and lead citrate (17,800, Electron Microscopy Sciences). Samples were visualized with an EM900 transmission electron microscope (Carl Zeiss).

Stritt S., Nurden P., Favier R., Favier M., Ferioli S., Gotru S.K., van Eeuwijk J.M., Schulze H., Nurden A.T., Lambert M.P., Turro E., Burger-Stritt S., Matsushita M., Mittermeier L., Ballerini P., Zierler S., Laffan M.A., Chubanov V., Gudermann T., Nieswandt B, & Braun A. (2016). Defects in TRPM7 channel function deregulate thrombopoiesis through altered cellular Mg2+ homeostasis and cytoskeletal architecture. Nature Communications, 7, 11097.

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Platelet Ultrastructure Analysis by EM

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Washed platelets in a concentration of 3 × 10⁵ platelets/μl were fixed with 2.5% glutaraldehyde (Electron Microscopy Science) in cacodylate buffer (pH 7.2, AppliChem). Epon 812 (Electron Microscopy Science) was used to embed platelets. After generation of ultrathin sections, platelets were stained with 2% uranyl acetate (Electron Microscopy Science) and lead citrate (Electron Microscopy Science). Sections were analyzed on a Zeiss EM900 electron microscope. Platinum replica electron microscopy of spread platelets was performed as previously described (28 (link)).

Baumann J., Sachs L., Otto O., Schoen I., Nestler P., Zaninetti C., Kenny M., Kranz R., von Eysmondt H., Rodriguez J., Schäffer T.E., Nagy Z., Greinacher A., Palankar R, & Bender M. (2022). Reduced platelet forces underlie impaired hemostasis in mouse models of MYH9-related disease. Science Advances, 8(20), eabn2627.

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Mitochondrial Dynamics and Apoptosis Assay

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Mito-Tracker Orange CM-H2TMRos (Molecular Probes, Cat No. M-7511) was supplied by Life Technologies. PI4KIIIα inhibitor, phenylarsine oxide (PAO, Sigma, Cat No. P-3075) and PI4KIIIβ inhibitor, PIK-93 (AdooQ BioScience, Cat No. A10731), were acquired from Sigma and AdooQ BioScience, respectively. Transfection reagents, polyethylenimine (PEI, Sigma Cat No. 408727) and GenJet in vitro DNA tranfection reagent for Huh-7 Cells (SignaGen, Cat No. SL100489-HUH) were purchased from Sigma and SignaGen, respectively. Hydrogen peroxide (Cat No. 1100066) was purchased from International Laboratory, USA). Apoptotic cells were detected by by TUNEL staining using In situ cell death detection kit, TMR red (Roche, Cat no. 12156792910).
For sample preparation for electron microscopy (EM), fixatives glutaldehyde, EM Grade (Electron Microscopy Sciences, Cat No. 16200) and osmium tetroxide (Electron Microscopy Sciences, Cat No. 19110); embedding medium, Embed-812 embedding kit (Electron Microscopy Sciences, Cat No. 14120); and stains, uranyl acetate (Electron Microscopy Sciences, Cat No. 22400) and lead citrate (Electron Microscopy Sciences, Cat No. 17800) were purchased from Electron Microscopy Sciences.

Siu G.K., Zhou F., Yu M.K., Zhang L., Wang T., Liang Y., Chen Y., Chan H.C, & Yu S. (2016). Hepatitis C virus NS5A protein cooperates with phosphatidylinositol 4-kinase IIIα to induce mitochondrial fragmentation. Scientific Reports, 6, 23464.

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Ultrastructural Analysis of Aged Mouse Cochlea

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The cochleae from different aged mice were isolated. Segments of the cochlear lateral wall from the basal turns were fixed overnight in phosphate-buffered 3% glutaraldehyde-1.5% paraformaldehyde and post-fixed in 1% osmium. Tissues were dehydrated through a graded alcohol series and embedded in Embed 812 (Electron Microscopy Sciences, Hatfield, PA), sectioned, stained with lead citrate (Electron Microscopy Sciences, Hatfield, PA) and uranyl acetate (Electron Microscopy Sciences, Hatfield, PA), and viewed on a Philips EM 100 transmission electron microscope (Philips/FEI Corporation, Eindhoven, Holland).

Neng L., Zhang J., Yang J., Zhang F., lopez I.A., Dong M, & Shi X. (2015). Structural changes in thestrial blood-labyrinth barrier of aged C57BL/6 mice. Cell and tissue research, 361(3), 685-696.

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Huh7 Cell Ultrastructure Analysis

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Following fixation of Huh7 cells in 2% glutaraldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) and 2% paraformaldehyde (EMS, USA) in 0.05 M sodium cacodylate (pH 7.2; Electron Microscopy Sciences) for 2 h at 4°C, specimens were fixed in 1% osmium tetroxide (Electron Microscopy Sciences) for 1 h at 4°C, dehydrated with increasing ethanol (25, 50, 70, 90 and 100%) for 5 min each at 4°C and embedded in epoxy resin (Embed 812; Electron Microscopy Sciences) for 48 h at 60°C according to the manufacturers' instructions. Ultrathin sections (60 nm) were prepared using an LKB-III ultratome (Leica Microsystems GmbH, Wetzlar, Germany) and were stained with 0.5% uranyl acetate (Electron Microscopy Sciences) for 20 min and 0.1% lead citrate (Electron Microscopy Sciences) for 7 min at room temperature. Images were recorded on a Hitachi H7650 electron microscope (magnification, ×10,000; Hitachi, Ltd., Tokyo, Japan) installed at the Center for University-Wide Research Facilities (CURF) at Chonbuk National University.

Nazim U.M, & Park S.Y. (2018). Attenuation of autophagy flux by 6-shogaol sensitizes human liver cancer cells to TRAIL-induced apoptosis via p53 and ROS. International Journal of Molecular Medicine, 43(2), 701-708.

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Ultrastructural Analysis of Anterior Commissure

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Animals at postnatal day 22 (P22) were euthanized with an overdose of Euthasol (Virbac) and immediately perfused transcardially with 1 mL/g of body weight of saline solution followed by ice-cooled 4% paraformaldehyde (PFA, JT Baker) and 0.2% glutaraldehyde (Electron Microscopy Sciences) in phosphate buffered saline (PBS). Brains were postfixed overnight at 4°C, and then 50 m coronal sections were collected with a vibratome (Pelco 101). Lipids were fixed by treating sections with 4% osmium (Electron Microscopy Sciences) for 1 h, dehydrated through graded alcohols, and treated with uranyl acetate (Electron Microscopy Sciences) and lead citrate (Electron Microscopy Sciences) to increase the contrast of tissue ultrastructure. Finally, sections were polymerized in EPON 812 (Electron Microscopy Sciences) and the AC was microdissected for sectioning at 0.07 m with an ultramicrotome (UltraCut-E). Sections were mounted on formvar coated slot grids (Electron Microscopy Sciences) before imaging.

Martin-Lopez E., Meller S.J, & Greer C.A. (2018). Development of Piriform Cortex Interhemispheric Connections Via the Anterior Commissure: Progressive and Regressive Strategies. Brain structure & function, 223(9), 4067-4085.

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