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Uranyless

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UranyLess is a positive staining solution for transmission electron microscopy (TEM) imaging. It is designed to enhance the contrast of samples, allowing for improved visualization of cellular structures and ultrastructural details.

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Lab products found in correlation

Lead citrate, by Electron Microscopy Sciences (4 mentions) Jem 1010 electron microscope, by JEOL (3 mentions) Jem 1400 flash tem, by JEOL (2 mentions) Morgagni 268d, by Philips (2 mentions) Axio imager, by Zeiss (2 mentions) 7700 transmission electron microscope, by Hitachi (2 mentions) 300 mesh formvar carbon coated copper grids, by Electron Microscopy Sciences (2 mentions) Copper grid, by Agar Scientific (1 mentions) Reichert ultracut s, by Leica camera (1 mentions) Epon 812, by Merck Group (1 mentions) Toluidine blue solution, by Merck Group (1 mentions) Osmium tetroxide, by Polysciences (1 mentions) Analysis software, by Olympus (1 mentions) Megaview 3 digital camera, by Olympus (1 mentions) Jem 1010 transmission electron microscope, by JEOL (1 mentions) Osmium tetroxide, by Electron Microscopy Sciences (1 mentions) Em109 electron microscope, by Zeiss (1 mentions) Axioskop 40, by Zeiss (1 mentions) Tecnai f20 tem, by Thermo Fisher Scientific (1 mentions) Lead citrate 3, by Electron Microscopy Sciences (1 mentions)

34 protocols using uranyless

1

Ultrastructural Analysis of Feline Skin Samples

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Skin punch biopsies from case no. 4 and the skin punch biopsy of an age-matched control cat were fixed in a 2.5% glutaraldehyde/0.1 M cacodylate buffer solution, then washed in 0.1 M cacodylate buffer (cacodylic acid sodium salt trihydrate; Merck KGaA, Darmstadt, Germany), and afterwards contrasted for 4 h in 1% osmium tetroxide (Polysciences Europe GmbH, Hirschberg, Germany). Samples were then dehydrated in an ascending series of ethanol and embedded in a mixture of Epon 812 (epoxy resin), dodecenylsuccinic anhydride (plasticizer), methylnadic anhydride (hardener), DMP 30 (catalyst) (all: Merck KGaA, Darmstadt, Germany), and polymerized at 60 °C for 5 days. Semi- and ultrathin sections were cut with an ultra-microtome Reichert Ultracut S (Leica, Wetzlar, Germany). Semithin sections (0.5 µm) were stained with 1% Toluidine blue solution (Merck KGaA, Darmstadt, Germany). From the semi-thin sections, representative areas for ultrastructural analysis were selected by light microscopy (Axioimager, Zeiss, Oberkochen, Germany). Ultrathin (80 nm) sections were mounted on copper-grids (Agar Scientific Ltd., Stansted, Essex, UK), contrasted with lead citrate and UranyLess (Electron Microscopy Sciences, Hatfield, PA, USA), and examined with a transmission electron microscope (CM12; Zeiss, Oberkochen, Germany).
Kiener S., Apostolopoulos N., Schissler J., Hass P.K., Leuthard F., Jagannathan V., Schuppisser C., Soto S., Welle M., Mayer U., Leeb T., Fischer N.M, & Kaessmeyer S. (2022). Independent COL5A1 Variants in Cats with Ehlers-Danlos Syndrome. Genes, 13(5), 797.
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2

Negative Staining Imaging of SMALPs

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SMALPs were prepared as described above to a final lipid concentration of 1 mM. 3% w/w SMA was added to both lipids before overnight equilibration. SMALPs of 14:1 PC were equilibrated with shaking at room temperature overnight, while 22:1 PC SMALPs were incubated at 60 C for the same duration. SMALPS were imaged with negative staining TEM. Small aliquots of SMALPs were adsorbed on to glow discharged carbon-coated copper EM grids (Electron Microscopy Sciences, Hatfield, PA) for 120 s. Grids were washed twice with ddH2O for 15 s before negative staining with UranyLess (Electron Microscopy Sciences, Hatfield, PA) for 45 s. Excess liquid was removed from the grid with filter paper between steps. Grids were air-dried prior to examination on a JEOL JEM 1400-Flash TEM (JEOL USA, Peabody, MA) operating at 80 kV. SMALPs were measured in ImageJ.
Stefanski K.M., Russell C., Westerfield J.M., Lamichhane R., & Barrera F.N. (2020). PIP2promotes conformation-specific dimerization of the EphA2 membrane region.
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3

TEM Visualization of Extracellular Vesicles

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Samples were prepared for transmission electron microscopy (TEM) by the negative and positive staining procedures. In brief, the EV mixtures comprising exosomes and ectosomes were fixed in Karnovsky’s fluid for 5 min at room temperature (RT), centrifuged for 5 min at 11,000× g, and then rinsed and resuspended in Cacodylate buffer 0.2 M. Aliquots of these suspensions were sedimented for 5 min at RT or 15 min at 37 °C on 300 mesh, and copper/formvar-coated grids depending on the kind of staining (respectively, negative and positive). The Uranyless (Electron Microscopy Sciences, Hatfield, PA, USA) was used both as negative and positive staining for 5 RT and 15 min at 37 °C. Dried samples were analyzed at a Jem 1010 transmission electron microscope (Jeol, Tokyo, Japan) at 80 Kv and photomicrographs acquired with a MegaView III digital camera (Soft Imaging System, Muenster, Germany) and analyzed with AnalySIS software (Soft Imaging System, Muenster, Germany).
Andreucci E., Ruzzolini J., Bianchini F., Versienti G., Biagioni A., Lulli M., Guasti D., Nardini P., Serratì S., Margheri F., Laurenzana A., Nediani C., Peppicelli S, & Calorini L. (2022). miR-214-Enriched Extracellular Vesicles Released by Acid-Adapted Melanoma Cells Promote Inflammatory Macrophage-Dependent Tumor Trans-Endothelial Migration. Cancers, 14(20), 5090.
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4

Ultrastructural Analysis of Subretinal Drusen Deposits

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To visualize the ultrastructure of SDD and membranes of cells surrounding the deposits, we prepared AMD tissue samples from prior studies for transmission electron microscopy. One block (Case 1, 76-year-old female) came from a histologic study of micro-dissected and pelleted drusen (47 (link)). Another block (Case 2, 88-year-old-male) came from macula-wide sections processed for the Project MACULA website (48 (link)). Both were preserved in 1% glutaraldehyde and 2.5% paraformaldehyde in phosphate buffer. To preserve extracellular lipids, samples were post-fixed with osmium tannic acid paraphenylenediamine (OTAP) (49 (link), 50 (link)). Tissue blocks from Cases 1–2 were embedded in epoxy resin (PolyBed 812, EMS, Hatfield PA) and re-sectioned for this study. Gold sections (nominally 90 nm thick) were mounted on grids (Formvar Carbon Support Film on Specimen Grid, Electron Microscopy Sciences, Fort Washington, PA) and post-stained with mixed lanthanides (UranyLess, Electron Microscopy Sciences, Fort Washington, PA). TEM images were acquired at original magnifications of up to 2100x (Tecnai 120kv TEM, FEI, Hillsboro, OR; BioSprint 29 Megapixel CCD camera, AMT, Woburn, MA). An electron micrograph of clinically documented SDD (Case 3) was made available from the Sarks Archive (Sydney Australia; Data Use Agreement 2021STE02369); this eye had been post-fixed in 2% osmium.
Anderson D.M., Kotnala A., Migas L.G., Patterson N.H., Tideman L., Cao D., Adhikari B., Messinger J.D., Ach T., Tortorella S., Van de Plas R., Curcio C.A, & Schey K.L. (2023). Lysolipids are prominent in subretinal drusenoid deposits, a high-risk phenotype in age-related macular degeneration. Frontiers in ophthalmology, 3, 1258734.
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5

Transmission Electron Microscopy Protocol

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Samples were processed by TEM, as previously reported [30 (link),31 (link)]. Briefly, pellets obtained by SCUF (Fn1 and Fn5) were carefully fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7) for 1 h at 4 °C. Samples were post-fixed with 1% osmium tetroxide (Electron Microscopy Sciences, Fort Washington, WA, USA) for 1 h at 4 °C, dehydrated with a graded acetone series (from 50% to 100%), and embedded in Spurr resin. Semithin sections were cut with a Powertome X RMC ultramicrotome (Science Services, Fort Washington, WA, USA), stained with 1% toluidine blue solution, and analyzed under a ZEISS Axioskop 40 (Carl Zeiss, Göttingen, Germany) light microscope, equipped with Coolsnap Videocamera (Photometrics, Tucson, AZ, USA). Ultrathin sections were stained with uranyless (Electron Microscopy Sciences, Fort Washington, WA, USA) and lead citrate. Samples were observed under a Zeiss EM109 electron microscope, and ultrastructural images were acquired with a GATAN Fastcam 830 CCD camera (Gatan Inc., Pleasanton, CA, USA).
Di Giuseppe F., Carluccio M., Zuccarini M., Giuliani P., Ricci-Vitiani L., Pallini R., De Sanctis P., Di Pietro R., Ciccarelli R, & Angelucci S. (2021). Proteomic Characterization of Two Extracellular Vesicle Subtypes Isolated from Human Glioblastoma Stem Cell Secretome by Sequential Centrifugal Ultrafiltration. Biomedicines, 9(2), 146.
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6

Visualizing α-Synuclein Structures

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Samples (50 μl) of a 1 mg/ml αS solution were adsorbed onto glow-discharged 200 mesh formvar/carbon coated copper grids (Electron Microscopy Sciences, Hatfield, PA) and stained with uranyLess (Electron Microscopy Sciences). Excess liquid was removed and grids were allowed to air dry. Samples were viewed and imaged using a Hitachi 7700 transmission electron microscope (Hitachi, Tokyo, Japan).
Acuña L., Hamadat S., Corbalán N.S., González-Lizárraga F., dos-Santos-Pereira M., Rocca J., Sepúlveda Díaz J., Del-Bel E., Papy-García D., Chehín R.N., Michel P.P, & Raisman-Vozari R. (2019). Rifampicin and Its Derivative Rifampicin Quinone Reduce Microglial Inflammatory Responses and Neurodegeneration Induced In Vitro by α-Synuclein Fibrillary Aggregates. Cells, 8(8), 776.
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7

Purification and Analysis of HBV Capsids

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HBV capsids were purified from AML12HBV10 cells, with or without treatment with 5 μM of BA-53038B for 2 days, by sucrose gradient centrifugation 53 (link).Electron microscopy analysis was performed after negatively stained with Uranyless (Electron Microscopy Sciences) on a FEI Tecnai 12 Spirit/Biotwin (LaB6 filament), operating at 100kV, with an AMT 2k × 2k CCD camera.
Zhang X., Cheng J., Ma J., Hu Z., Wu S., Hwang N., Kulp J., Du Y., Guo J.T, & Chang J. (2018). Discovery of novel hepatitis B virus nucleocapsid assembly inhibitors. ACS infectious diseases, 5(5), 759-768.
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8

Nanoparticle F TEM Imaging

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TEM images of Nanoparticle F were taken by adding a small drop (1–2μL) of sample on to a copper grid with a carbon film. Prior to loading the samples, the grid was modified by glow discharge treatment using Quorum GloQube to make the carbon film hydrophilic. The sample was loaded onto the grid, dried, and stained with UranyLess (Electron Microscopy Sciences) and Lead Citrate 3% (Electron Microscopy Sciences). The grids were then imaged using a FEI Tecnai F20 TEM.
De La Torre A.L., Smith C., Granger J., Anderson F.L., Harned T.C., Havrda M.C., Chang C.C, & Chang T.Y. (2021). Facile method to incorporate high-affinity ACAT/SOAT1 inhibitor F12511 into stealth liposome-based nanoparticle and demonstration of its efficacy in blocking cholesteryl ester biosynthesis without overt toxicity in neuronal cell culture. Journal of neuroscience methods, 367, 109437.
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9

Ultrastructural Localization of VGLUT3

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Lumbar or sacral mouse or rat spinal cord sections were used for preembedding immunoperoxidase labeling of VGLUT3. Mouse tissue sections were initially incubated in 1% NaBH4 in PBS for 30 min to quench free aldehyde groups. After permeabilization in 50% ethanol for 30 min and incubation in PBS with 1% BSA for 1 h, the sections were incubated in primary antibody solution (mouse anti-VGLUT3, 1:1000 in PBS with 1% BSA) at room temperature overnight or for 72 h. After rinsing, the sections were subsequently incubated in biotinylated goat anti-mouse secondary antibody (Vector Laboratories) in PBS with 1% BSA and in Vector ABC (Vector Laboratories) for 2–3 h each. Peroxidase activity was visualized by incubation in ImmPACT DAB (Vector Laboratories) for 30 s to 2 min. Sections thus labeled for VGLUT3 were rinsed briefly in PB (0.1 M; pH 7.4), incubated in 0.5–1% OsO4 in PB for 10–30 min (depending on section thickness), dehydrated in graded series of ethanol and embedded in Durcupan (Electron Microscopy Sciences). Ultrathin sections of embedded tissue were counterstained using 2% uranyl acetate (15 min) or UranyLess (2 min; Electron Microscopy Sciences) followed by 0.4% lead citrate before examination in a JEOL 1230 electron microscope.
Larsson M, & Broman J. (2019). Synaptic Organization of VGLUT3 Expressing Low-Threshold Mechanosensitive C Fiber Terminals in the Rodent Spinal Cord. eNeuro, 6(1), ENEURO.0007-19.2019.
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10

Structural Analysis of SMALPs

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SMALPs were prepared as described above to a final lipid concentration of 1 mM. In total, 3% w/w SMA was added to both lipids before overnight equilibration. SMALPs of 14:1 PC were equilibrated with shaking at room temperature (19–21 °C) overnight, while 22:1 PC SMALPs were incubated at 60 °C for the same duration. SMALPS were imaged with negative staining TEM. Small aliquots of SMALPs were adsorbed on to glow discharged carbon-coated copper EM grids (Electron Microscopy Sciences, Hatfield, PA) for 120 s. Grids were washed twice with ddH2O for 15 s before negative staining with UranyLess (Electron Microscopy Sciences, Hatfield, PA) for 45 s. Excess liquid was removed from the grid with filter paper between steps. Grids were air-dried prior to examination on a JEOL JEM 1400-Flash TEM (JEOL USA, Peabody, MA) operating at 80 kV. SMALPs were measured in ImageJ.
Stefanski K.M., Russell C.M., Westerfield J.M., Lamichhane R, & Barrera F.N. (2020). PIP2 promotes conformation-specific dimerization of the EphA2 membrane region. The Journal of Biological Chemistry, 296, 100149.
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