Uranyl acetate is a chemical compound with the formula UO2(CH3COO)2.
It is commonly used as a staining agent in electron microscopy to enhance contrast of biological samples.
Uranyl acetate can also be utilized in the synthesis of other uranium compounds and as a precursor in the production of uranium-based catalysts.
This versatile chemical has applications in various fields, including materials science, nuclear technology, and biochemistry.
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Postnatal day 30 (P30) TWI mice and their WT littermates (5 for each experimental group processed in 5 different experimental sessions, every TWI with its WT littermate) and one P15 TWI mouse versus its WT littermate were perfused with a fixative solution (4% paraformaldehyde and 0.1%–1%–2.5% glutaraldehyde in phosphate buffer, pH 7.4). Sciatic nerves, spinal cords and gastrocnemius muscles were dissected and post-fixed for 4 hours at room temperature in the same fixative solution. Spinal cords were dissected in the lumbar region, isolating four 1-mm-thick sections in the lumbar enlargement region and the gastrocnemius muscles were cut in small portions, approximately 1 mm3 in volume. Sciatic nerves were processed without further sectioning. The selected tissues were further treated for epoxy resin embedding as previously described43 . Briefly, the samples were deeper fixed in 2–2.5% glutaraldehyde in cacodylate buffer (0.1 M, pH 7.4). After rinsing, specimens were post-fixed with osmium tetroxide (1%)-potassium ferricyanide (1%) in cacodylate buffer, rinsed again, en bloc stained with 3% uranyl acetate in ethanol, dehydrated and embedded in epoxy resin, that was baked for 48 h at 60 °C. Thin sections were obtained with an ultramicrotome (UC7, Leica Microsystems, Vienna, Austria) and collected on G300Cu grids (EMS). Finally, sections were examined with a Zeiss LIBRA 120 plus transmission electron microscope equipped with an in-column omega filter.
Cappello V., Marchetti L., Parlanti P., Landi S., Tonazzini I., Cecchini M., Piazza V, & Gemmi M. (2016). Ultrastructural Characterization of the Lower Motor System in a Mouse Model of Krabbe Disease. Scientific Reports, 6, 1.
Full details of experimental procedures are provided in the supplementary materials. SpyCas9 and its point mutants were expressed in Escherichia coli Rosetta 2 strain and purified essentially as described (8 (link)). SpyCas9 crystals were grown using the hanging drop vapor diffusion method from 0.1 M tris-Cl (pH 8.5), 0.2 to 0.3 M Li2SO4, and 14 to 15% (w/v) PEG 3350 (polyethylene glycol, molecular weight 3350) at 20°C. Diffraction data were measured at beamlines 8.2.1 and 8.2.2 of the Advanced Light Source (Lawrence Berkeley National Laboratory), and at beamlines PXI and PXIII of the Swiss Light Source (Paul Scherrer Institut) and processed using XDS (50 (link)). Phasing was performed with crystals of selenomethionine (SeMet)–substituted SpyCas9 and native Cas9 crystals soaked individually with 10 mM Na2WO4, 10 mM CoCl2, 1 mM thimerosal, and 1 mM Er(III) acetate. Phases were calculated using autoSHARP (51 (link)) and improved by density modification using Resolve (52 (link)). The atomic model was built in Coot (53 (link)) and refined using phenix.refine (54 (link)). A. naeslundii Cas9 (AnaCas9) was expressed in E. coli Rosetta 2 (DE3) as a fusion protein containing an N-terminal His10 tag followed by MBP and a TEV (tobacco etch virus) protease cleavage site. The protein was purified by Ni-NTA (nickel–nitrilotriacetic acid) and heparin affinity chromatography, followed by a gel filtration step. Crystals of native and SeMet-substituted AnaCas9 were grown from 10% (w/v) PEG 8000, 0.25 M calcium acetate, 50 mM magnesium acetate, and 5 mM spermidine. Native and SeMet single-wavelength anomalous diffraction (SAD) data sets were collected at beamline 8.3.1 of the Advanced Light Source, processed using Mosflm (55 (link)), and scaled in Scala (56 (link)). Phases were calculated in Solve/Resolve (52 (link)), and the atomic model was built in Coot and refined in Refmac (57 (link)) and phenix.refine (54 (link)). For biochemical assays, crRNAs were synthesized by Integrated DNA Technologies, and tracrRNA was prepared by in vitro transcription as described (8 (link)). The sequences of RNA and DNA reagents used in this study are listed in table S2. Cleavage reactions were performed at room temperature in reaction buffer [20 mM tris-Cl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 5% glycerol, 1 mM dithiothreitol] using 1 nM radio-labeled dsDNA substrates and 1 nM or 10 nM Cas9:crRNA:tracrRNA. Cleavage products were resolved by 10% denaturing (7 M urea) PAGE and visualized by phosphorimaging. Cross-linked peptide-DNA heteroconjugates were obtained by incubating 200 pmol of catalytically inactive (D10A/H840A) Cas9 with crRNA:tracrRNA guide and 10-fold molar excess of BrdU containing dsDNA substrate for 30 min at room temperature, followed by irradiation with UV light (308 nm) for 30 min. S1 nuclease and phosphatase–treated tryptic digests were analyzed using a Dionex UltiMate3000 RSLCnano liquid chromatograph connected in-line with an LTQ Orbitrap XL mass spectrometer equipped with a nanoelectrospray ionization source (Thermo Fisher Scientific). For negative-stain EM, apo-SpyCas9, SpyCas9: RNA, and SpyCas9:RNA:DNA complexes were reconstituted in reaction buffer, diluted to a concentration of ~25 to 60 nM, applied to glow-discharged 400-mesh continuous carbon grids, and stained with 2% (w/v) uranyl acetate solution. Data were acquired using a Tecnai F20 Twin transmission electron microscope operated at 120 keV at a nominal magnification of either ×80,000 (1.45 Å at the specimen level) or ×100,000 (1.08 Å at the specimen level) using low-dose exposures (~20 e− Å−2) with a randomly set defocus ranging from −0.5 to −1.3 μm. A total of 300 to 400 images of each Cas9 sample were automatically recorded on a Gatan 4k × 4k CCD (charge-coupled device) camera using the MSI-Raster application within the automated macromolecular microscopy software Leginon (58 (link)). Particles were preprocessed in Appion (45 (link)) before projection matching refinement with libraries from EMAN2 and SPARX (59 (link), 60 (link)) using RCT reconstructions (34 (link)) as initial models.
Jinek M., Jiang F., Taylor D.W., Sternberg S.H., Kaya E., Ma E., Anders C., Hauer M., Zhou K., Lin S., Kaplan M., Iavarone A.T., Charpentier E., Nogales E, & Doudna J.A. (2014). Structures of Cas9 Endonucleases Reveal RNA-Mediated Conformational Activation. Science (New York, N.Y.), 343(6176), 1247997.
We performed two-photon imaging in the mouse visual cortex as described previously27 (link),30 (link) by recording calcium responses to visual stimuli consisting of drifting gratings in each of 16 directions. We then acquired an in vivo fluorescent anatomical volume after injecting the tail vein with SR101 (100 mM) to label vasculature. The animal was perfused transcardially (2% paraformaldehyde/2.5% glutaraldehyde) and the brain was processed for serial-section TEM. Serial thin (<50 nm) sections were cut, picked up on pioloform-coated slot grids, and then post-stained with uranyl acetate and lead citrate. 1,215 serial sections were imaged at 120 kV on a JEOL 1200 EX with a custom scintillator atop optical-quality leaded vacuum glass at the end of a custom-built vacuum chamber extension. Custom software controlled automated x–y stage motion and image acquisition with a 2x2 array of CCD cameras (Imperx IPX-11M5) and Zeiss lenses. Images suitable for circuit reconstruction were acquired at a net rate of 5–8 MPix/s. Camera images were aligned in 2–D by registering adjacent camera images and dewarping, followed by histogram equalization and stitching. Then adjacent sections were registered and 3-D deformations were equalized in aligning the EM volume. Axonal and dendritic arbours of the functionally characterized neurons were manually reconstructed using TrakEM2 and objects were classified using classical criteria33 . Neurons or dendritic fragments receiving synapses from multiple functionally characterized cells were included in analysis of convergence. For each synapse participating in a convergence, a second individual (blind to the original reconstruction) traced the pre- and the post-synaptic processes, starting from the synapse. Segmentation that diverged between the two tracers was excluded from further analysis. Cumulative synaptic proximity (CSP) of pairs of axons was calculated by centring a 3-D Gaussian density function at each synaptic bouton and taking the sum of their dot products over all pairs of synapses.
Bock D.D., Lee W.C., Kerlin A.M., Andermann M.L., Hood G., Wetzel A.W., Yurgenson S., Soucy E.R., Kim H.S, & Reid R.C. (2011). Network anatomy and in vivo physiology of visual cortical neurons. Nature, 471(7337), 177-182.
Yeast Strains and Constructs—The following yeast strains were used: BY4741 (MATa his3Δ1 leu2Δ met15Δ ura3Δ) and NDY257 (BY4741 rtn1::kanMX4 rtn2::kanMX4 yop1::kanMX) (6 (link)). Strains expressing GFP fusions to the chromosomal alleles of YOP1 and RTN1 were obtained from Invitrogen. The plasmid encoding Sec63-GFP (pJK59) has been previously described (12 (link)). To make the plasmid encoding Rtn1-GFP (pCV19), the SEC63 portion of pJK59 was removed by digestion with XbaI and XhoI. The RTN1 gene, including 400 bp upstream of the start site, was PCR-amplified from yeast chromosomal DNA and inserted into the same sites. Mammalian Plasmid Constructs—HA-DP1 was described previously (6 (link)). HA-Rtn3c was cloned by PCR amplifying Rtn3c (NCBI accession number: BC036717) from mouse cDNA with primers containing an N-terminal HA tag and inserted into pcDNA3.1D (Invitrogen). For Rtn4a-GFP, human Rtn4a was PCR-amplified from Rtn4a-Myc (described in a previous study (6 (link))) and ligated into the pAcGFP-N1 backbone (Clontech) using the XhoI and KpnI restriction sites at the 5′ and 3′ ends, respectively. For GFP-Rtn3c, Rtn3c was PCR-amplified from HA-Rtn3c and ligated into the pAcGFP-C1 backbone (Clontech) using the XhoI and EcoRI restriction sites. To clone GFP-Rtn4HD, the region encoding amino acids 961–1192 was PCR-amplified from human Rtn4a-Myc and inserted into pAcGFP-C1 using the XhoI/EcoRI restriction sites. GFP-DP1 was subcloned by PCR-amplifying mouse DP1 from HA-DP1 (described in a previous study (6 (link))) and inserting into pAc-GFP C1 using SacI/BamHI restriction sites. For GFP-Climp63, Climp63 was PCR-amplified from mouse cDNA and cloned into pAcGFP-C1 using the XhoI/EcoRI sites. Climp63Δlum-GFP was cloned by PCR amplifying the region encoding amino acids 1–115 (as described in (13 (link))) from GFP-Climp63 and inserted into pAcGFP-N1 using XhoI/EcoRI restriction sites. LBR-GFP was PCR-amplified from plasmid containing human LBR (14 (link)) and cloned into pAcGFP-N1 using the XhoI/BamHI restriction sites. For GFP-Sec61β, human Sec61β was PCR-amplified from the pcDNA3.1/GFP-Sec61β construct described previously (6 (link)), and inserted into pAcGFP-C1 using the BglII/EcoRI restriction sites. RFP-Sec61β was subcloned from GFP-Sec61β using the same restriction sites as above and inserted into an mRFP1 vector (pEGFP-C1 vector backbone where pEGFP has been replaced with mRFP1). Microscopy of Yeast—Yeast strains were grown in synthetic complete medium (0.67% yeast nitrogen base and 2% glucose) and imaged live at room temperature using an Olympus BX61 microscope, UPlanApo 100×/1.35 lens, QImaging Retiga EX camera, and IPlabs version 3.6.4 software. Screen for Mutations in Yeast RTN1 That Affect Localization—Error-prone PCR on RTN1 was performed using the GeneMorphII Random Mutagenesis Kit (Stratagene). The product of this reaction and pJK59 cut with XbaI and XhoI were used to transform wild-type yeast. Transformants were visually screened for those that showed perinuclear GFP localization. Tissue Culture, Indirect Immunofluorescence, and Confocal Microscopy of COS-7 Cells—Cells were grown at 37 °C with 5% CO2 in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and subcultured every 2–3 days. Transfection of DNA into cells was performed using Lipofectamine 2000 (Invitrogen). After 5 h of transfection, cells were split onto acid-washed No. 1 coverslips and allowed to spread for an additional 24–36 h before being processed for indirect immunofluorescence. For immunofluorescence, transfected cells were fixed in PBS containing 4% paraformaldehyde (Electron Microscopy Sciences) for 15 min, washed twice, and permeabilized in 0.1% Triton X-100 (Pierce) in PBS for 5–15 min. Cells were washed twice again and then probed with primary antibodies for 45 min in PBS containing 1% calf serum, at the following concentrations: rat anti-HA antibody (Roche Applied Science) at 1:200 dilution; mouse anti-αtubulin (Sigma) at 1:500 dilution; and rabbit anti-calreticulin antibody (Abcam) at 1:500 dilution. Cells were washed three times in PBS, and then incubated with various fluorophore-conjugated secondary antibodies for an additional 45 min (Alexafluor 488 or 555 anti-mouse at 1:250 dilution, Alexafluor 647 anti-rabbit 1:500 dilution, and Alexafluor 488 anti-rat 1:200 dilution (all from Invitrogen)). Cells were then washed and mounted onto slides using Fluoromount-G mounting medium (Southern Biotech). All imaging for indirect immunofluorescence was captured using a Yokogawa spinning disk confocal on a Nikon TE2000U inverted microscope with a 100× Plan Apo numerical aperture 1.4 objective lens, and acquired with a Hamamatsu ORCA ER cooled charge-coupled device camera using MetaMorph 7.0 software. For image presentation, brightness and contrast were adjusted across the entire image using Adobe Photoshop 7.0, and images were converted from 12 to 8 bits. Transmission Electron Microscopy—COS-7 cells expressing GFP-Rtn4HD were sorted in a MoFlo cell sorter (Cytomation). The resulting cell pellet was fixed for 1 h in a mixture of 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 m sodium cacodylate buffer (pH 7.4), washed in 0.1 m cacodylate buffer, and postfixed with a mixture of 1% OsO4 and 1.5% KFeCN6 for 30 min. The pellet was then washed in water and stained in 1% aqueous uranyl acetate for 30 min followed by dehydration in grades of alcohol (50%, 70%, and 95%, 2 × 100%). Next, the pellet was infiltrated in a 1:1 mixture of propylene oxide and TAAB Peon (Maria Canada Inc.) for 2 h, placed in pure TAAB Epon in a silicon-embedding mold, and polymerized at 65 °C for 48 h. Ultrathin sections (∼60–80 nm) were cut on a Reichert Ultracut-S microtome, placed onto copper grids, and stained with 0.2% lead citrate. Specimens were examined on a Tecnai G Spirit BioTWIN transmission electron microscope, and images were acquired with a 2k AMT charge-coupled device camera. Fluorescence Recovery after Photobleaching—Transfected COS-7 cells were imaged in phenol red-free HyQ DME (HyClone) supplemented with 25 mm Hepes, pH 7.4, and 1% fetal bovine serum. FRAP experiments were conducted on a Zeiss LSM 510 NLO laser scanning inverted microscope using a Plan-Neofluor 100×/1.3 oil objective with argon laser line 488 nm (optical slices <1.2 mm for COS-7 and 4.2 μm for yeast). Mammalian cell experiments were done at 37 °C using an objective heater (Bioptechs) and an enclosed stage incubator (Zeiss). LSM 510 software version 3.2 was used for image acquisition and analysis. Magnification, laser power, and detector gains were identical across samples. For all mammalian experiments, COS-7 cells were treated with 0.5 μm nocodazole, and all data were collected during the first 5–30 min of nocodazole addition. For photobleaching all constructs, except for LBR-GFP, the tubular ER was magnified using the 3× zoom function so that individual tubules could be seen clearly. For LBR-GFP, the microscope was focused onto the bottom of the nuclear envelope. Images taken for 5-s prebleaching, whereupon a region of interest of 65 × 65 pixels was photobleached at 100% laser power. After the photobleaching, images were taken at 1-s intervals for 75–300 s. Yeast cells were treated similarly except that the region of interest was 17 × 17 pixels, and images were taken every 2–4 s at room temperature. Raw data were quantitated using Zeiss LSM510Meta software. For analysis, the fluorescence intensity of three regions of interest was measured: the photobleached region (PR), a region outside of the cell to check for overall background fluorescence (BR), and a region within the cell that was not photobleached to check for overall photobleaching and fluorescence variation (CR), for the entire course of the experiment. Microsoft Excel was used to normalize the relative fluorescence intensity, I, for each individual FRAP experiment using Equation 1. For data presentation, the mean averages of the normalized data for each set of FRAP experiments were plotted using GraphPad Prism 5.0, and fluorescence recovery curves were shown for the first 80–140 s of each experiment. Estimated half-times of recovery and mobile fraction values were calculated using the standard Michaelis-Menten equation. Sucrose Gradient Centrifugation—For yeast sucrose gradient analysis, crude membranes were isolated from yeast strains expressing GFP-fused proteins at endogenous levels as follows: 200 ml of culture were grown to OD ∼1, pelleted and then resuspended in TKMG lysis buffer (50 mm Tris, pH 7.0, 150 mm KCl, 2 mm MgCl2, 10% glycerol, 1 mm EDTA, 1 mm PMSF, 1 mm 4-(2-aminoethyl)benzenesulfonylfluoride hydrochloride), flash frozen in liquid nitrogen, and ground using a mortar and pestle. Cell debris was separated from the lysate by low speed centrifugation for 5 min at ∼2,000 × g. Membranes were then pelleted by ultracentrifugation for 15 min at 100,000 × g and solubilized in 200 μl of TKMG buffer containing 1% digitonin. Solubilized lysate was centrifuged for 10 min at 12,000 × g to separate out any remaining cell debris. 100-μl of lysate were run on 5–30% w/v sucrose gradients for 4 h at 166,000 × g at 25 °C on a Beckman TLS55 rotor. Twenty gradient fractions were collected from top to bottom and analyzed by SDS-PAGE and immunoblotting with anti-GFP antibody (Roche Applied Science). 50 mg of apoferritin, catalase, and aldolase was used as molecular weight standards. Xenopus washed membrane fractions were prepared in MWB (50 mm Hepes, pH 7.5, 2.5 mm MgCl2, 250 mm sucrose, and 150 mm potassium acetate) as previously described (6 (link)), incubated for 60 min at 25 °C in MWB containing 200 mm KCl and 0.5 mm GTP, and then solubilized for 30 min at 25 °C with either 2% Nonidet P-40 or 1.25% digitonin. Samples were pelleted for 15 min at 12,000 rpm, and the soluble fraction was loaded onto a 10–30% w/v sucrose gradient made with MWB containing 200 mm KCl, 0.1 mm GTP, and either 0.1% Nonidet P-40 or 0.1% digitonin, respectively. The sucrose gradient was centrifuged for 3 h, 45 min at 55,000 rpm. Sixteen gradient fractions were collected and analyzed by SDS-PAGE and immunoblotted with antibody against Xenopus Rtn4 (described in a previous study (6 (link))). For mammalian sucrose gradient analysis, COS-7 cells transiently transfected with HA-DP1 or GFP-Sec61β were harvested by scraping and then lysed and solubilized in HKME buffer (25 mm Hepes, pH 7.8, 150 mm potassium acetate, 2.5 mm magnesium acetate, 1 mm EDTA, and 2 mm PMSF) containing 1% digitonin for 1 h. The lysate was clarified by centrifugation at 10,000 × g for 10 min, and 100 μl of clarified lysate was sedimented on 5–30% w/v sucrose gradients under the same conditions as yeast. Fractions were analyzed by SDS-PAGE and immunoblotting with anti-HA antibody or anti-Sec61β antibody (described in a previous study (15 (link))). Chemical Cross-linking Experiments—Yeast crude membrane fractions were resuspended in buffer containing 50 mm Hepes, pH 7.0, 150 mm KCl, and 1 mm PMSF. Ethylene glycobis(succinimidylsuccinate) (EGS, Pierce), was dissolved in anhydrous DMSO and diluted to the desired concentration. 1 μl of EGS was added into every 20 μl of protein-containing sample for 30 min at room temperature. The reactions were quenched for 15 min with 2 μl of 1 m Tris, pH 7.5. Samples were analyzed on a 4–20% SDS-PAGE and immunoblotted using standard procedures with mouse anti-His or rat anti-HA antibody conjugated to peroxidase (Sigma). For mammalian cross-linking experiments, transfected COS-7 cells were grown in a 10-cm plate to ∼80% confluency and then lysed using a standard hypotonic lysis protocol. Briefly, cells were harvested in PBS, washed, incubated in hypotonic buffer (10 mm Hepes, pH 7.8, 10 mm potassium acetate, 1.5 mm magnesium acetate, 2 mm PMSF) for 10 min, and then passed through a 25-gauge syringe ten times. Nuclei and any remaining intact cells were separated from the lysate by centrifugation for 5 min at 3,000 × g, and the supernatant was then centrifuged for 10 min at 100,000 × g to pellet the membrane fraction. The membrane pellet was washed in HKM buffer (25 mm Hepes pH 7.8, 150 mm potassium acetate, 2.5 mm magnesium acetate, and 2 mm PMSF), repelleted at 100,000 × g, and resuspended to a final volume of 60 μl in HKM buffer. 10-μl membrane aliquots were used for each cross-linking reaction using the same conditions as above. Samples were analyzed on a 4–20% SDS-PAGE and immunoblotted using standard procedures with anti-HA antibody.
Rtn1p and Yop1p have slow diffusional mobility in the ER of yeast cells.A, typical FRAP of Sec63-GFP or Rtn1-GFP in S. cerevisiae cells expressed at endogenous levels. Images were taken before and then after the photobleach for the times indicated. The boxed region shows the area that was photobleached. B, fluorescence intensities normalized to prebleach values of FRAP analyses on yeast Sec63-GFP, Rtn1-GFP, and Yop1-GFP were plotted over time. Error bars indicate ± S.E.; n = 4 cells. C, fluorescence intensities normalized to prebleach values plotted over time of FRAP analyses on yeast Rtn1p in ATP-depleted (green) or non-depleted (orange) cells, compared with that of Sec63p-GFP (ATP depleted in blue; non-depleted in red). Error bars indicate ± S.E., n = 4 cells.
ATP Depletion Experiments—For yeast experiments, ATP was depleted by the addition of 10 mm 2-deoxy-d-glucose and 10 mm sodium azide (both from Sigma) for 2–5 min, and FRAP experiments were performed using the same parameters as described above. Similarly, for mammalian cell experiments, COS-7 cells were depleted of ATP as follows: transfected cells were washed twice in Opti-Mem serum-free media (Invitrogen) and then incubated with 50 mm 2-deoxy-d-glucose and 0.02% sodium azide in glucose-free imaging buffer (50 mm Hepes, pH 7.4, 150 mm potassium acetate, 2.5 mm magnesium acetate, and 1% fetal bovine serum). FRAP experiments were conducted in the same medium and completed within 5–30 min of treatment using the same parameters as above.
Shibata Y., Voss C., Rist J.M., Hu J., Rapoport T.A., Prinz W.A, & Voeltz G.K. (2008). The Reticulon and Dp1/Yop1p Proteins Form Immobile Oligomers in the Tubular Endoplasmic Reticulum. The Journal of Biological Chemistry, 283(27), 18892-18904.
Ethics declarations - All methods were performed in accordance with relevant guidelines and regulations. This work was approved by the Ethics Committee on Research with Humans from the Institute of Biomedical Sciences, University of São Paulo, Brazil (permission number 74683917.1.0000.5467). All specimens were handled under the Laboratory biosafety guidance required for the novel coronavirus (2019-nCoV) by the World Health Organization (WHO)13 at BLS3 facilities at the Institute of Biomedical Sciences, University of São Paulo. Clinical specimen collection - Nasopharyngeal (NP) swab samples were collected from symptomatic patients who had acquired COVID-19 during travels to northwest of Italy (Lombardia region) and returned to the São Paulo city in late February. These patients were treated in the same hospital and were the two first confirmed cases of COVID-19 in the São Paulo city. The specimens were collected on day 2-4 post-symptom onset, placed in 1-2 mL of saline medium and used for molecular diagnosis and virus isolation. Nucleic acid extraction and real-time RT-qPCR for virus detection - In order to perform the identification of SARS-CoV-2, the extraction of total nucleic acid (RNA and DNA) from the collected samples (200 µL of initial material) were carried out using the semi-automated NucliSENS® easyMag® platform (bioMérieux, Lyon, France), following the manufacturer’s’ instructions. All specimens were handled under the laboratory biosafety guidance required for the novel coronavirus (2019-nCoV) by WHO13 at BLS3 facilities at the Institute of Biomedical Sciences, University of São Paulo. The detection of viral RNA was carried out using the AgPath-ID One-Step RT-PCR Kit (Applied Biosystems Inc., Waltham, USA) on an ABI 7500 SDS real-time PCR machine (Applied Biosystems, Weiterstadt, Germany), using a published protocol and sequence of primers and probe for E gene.14 RNA copies/mL was quantified by real-time RT-qPCR using a specific in vitro-transcribed RNA quantification standard, kindly granted by Christian Drosten, Charité - Universitätsmedizin Berlin, Germany, as described previously.2Virus isolation - We used Vero E6 cells for isolation and initial passages. We cultured Vero E6 in Dulbecco minimal essential medium (DMEM) supplemented with 10% of heat-inactivated foetal bovine serum (FBS) (Vitrocell Embriolife, Campinas, Brazil). We used NP swab specimen for virus isolation. For isolation and first passage, we sow cells in a 25 cm2 cell culture flask in a concentration of 5 × 105 cells/mL. After 24 h, we removed the culture medium, washed three times with FBS free-DMEM and inoculated aliquots (500 μL) of the clinical specimens into the flask. After 1 h of incubation (adsorption), we completed the volume for 5 mL with DMEM supplemented with 2.5% FBS and 1% of penicillin-streptomycin. We grew the inoculated cultures in a humidified 37°C incubator in an atmosphere of 5% CO2 and observed for cytopathic effects (CPE) daily up to 72 h. Supernatant was collected daily, and virus replication was confirmed through CPE, gene detection and electron microscopy. Virus titration - Median tissue culture infectious dose (TCID50/mL) - Vero E6 and CCL-81 cells were seeded into 96-well plate (5 × 104 cells/mL), 24 h before the experiment. Virus was 10-fold serially diluted in medium (10-1 to 10-12). Medium was removed from plates, virus dilutions applied in sextuplicate and incubated at 37°C. Visualisations were performed daily in an inverted light microscope (Axiovert 100, Carl Zeiss Oberkochen) to observe the CPE. After 72 h, the last reading was performed, and the monolayers were fixed and stained with Naphthol Blue Black (Sigma-Aldrich Co., Deisenhofen, Germany) dissolved in sodium acetate-acid acetic. The viral titre was expressed in TCID50/mL and calculated using the Spearman & Kärber algorithm, as described by Hierholzer & Killington.15Plaque forming units (PFU/mL) - Virus titration was carried out in 24 wells plates seeded with Vero E6 and CCL-81 cells at a concentration of 1 × 105 cells/well. After 24 h and a cell confluence of 80-90%, dilutions 10-1 to 10-10 in DMEM 2.5% FBS of the virus was transferred in duplicate (100 µL/well) to the seeded plates. After 1 h adsorption at 37oC 5% CO2, the wells were completed with an overlay of carboxymethyl cellulose (CMC) with DMEM, 2% FBS and 1% of penicillin-streptomycin, and plates incubated at 37oC in 5% CO2 and stained with Naphtol Blue Black dissolved in sodium acetate-acid acetic. Plates were observed and stained from 48 to 96 h post-inoculation (h.p.i.). Both virus titration (TCID50/mL and PFU/mL) were made after the third passage of the isolated virus (T2). Negative stain transmission electron microscopy - Samples were adsorbed to glow-discharged carbon film-coated copper grids (400 Mesh, CF400-Cu, Electron Microscopy Sciences). The grids were washed with ultrapure water treated with DEPC and negatively stained with uranyl acetate 2% (w/v) with blotting on filter paper after each step. A FEI Tecnai G20 200 kV transmission electron microscope (Department of Cell and Developmental Biology, Institute of Biomedical Sciences, University of São Paulo) was used for image acquisition. Virus growth kinetics in different cell lines - Three Vero cell lines (E6, CCL-81 and hSLAM) plus a human epithelial type 2 (HEp-2) cells, at concentration of 5 × 104 cells/mL, were tested for the propagation of the SARS-CoV-2 by inoculation at a multiplicity of infection (MOI) of 0.02. The culture medium consisted of DMEM supplemented with 2.5% of FBS. Aliquots of cell-associated and supernatants compartments were collected every 12 h up to 96 h.p.i. for virus quantification via TCID50/mL and RNA copy number quantification by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The assay was conducted in triplicate, reproduced in two independent experiments and expressed by standard error of the mean (SEM). Graphics and SEM were done using GraphPad Prism software version 8.1 (GraphPad Software, San Diego, USA). Next generation sequencing of viral full-length genome - We extracted total nucleic acid from the NP and oropharyngeal (OP) swab samples and cell supernatants isolates with the QIAamp Viral RNA Mini kit (QIAGEN, Hilden, Germany). The purification and concentration steps were carried out with RNA Clean & Concentrator kit (Zymo Research, Irvine, USA) with DNAse I treatment during the concentration process. Depletion of human ribosomal RNA was performed with the concentrated RNA product using the QIAseq Fast Select RNA Removal kit (QIAGEN). Finally, the RNA samples were submitted to random amplification following the methodology described in Greninger et al.16 with few modifications. The preparation of sequencing libraries for the Illumina platform was carried out with the Nextera XT Kit (Illumina, San Diego, USA) and multiplex testing, using the random two-step PCR amplification product as input, followed the kit’s standard instructions. The libraries were quantified after fluorescence measuring with the Qubit instrument (Thermo Fisher Scientific, Waltham, USA) and loaded on the NextSeq 550 equipment (Illumina) for sequencing with MID 300 paired-end reads (Illumina). Sequencing analysis - The sequencing data was analysed by a flow of bioinformatics analysis (pipeline) developed at Albert Einstein Hospital. In summary, raw sequencing data was subjected to sequence quality controls, removal of human contaminants by aligning against the HG19 reference genome, taxonomic identification of other pathogens and genome recovery through manual curing. Quality control was performed using cutadapt17 to filter sequences by length (< 50 bp), average quality (Qp < 20) and trim options to remove low quality ends (9 bp to 5’ end and 5 bp to 3’ end). Passed QC reads were mapped to HG19 human reference genome using bwa18 mem with default parameters. Not mapped reads were submitted to assembly using SPADES 1.13.19 Contigs were inspected and manually curated using Geneious 2020.1 to generate a final assembly. Complete genome was compared to SARS-CoV-2 reference and close isolates by multiple sequence alignment. Final genome was deposited in GenBank (https://www.ncbi.nlm.nih.gov/genbank/). Production of virus isolates (VIS) and lysate (VLS) stocks and national distribution network - The preparation of VIS and VLS stocks was performed as described above for virus isolation. Related to VIS, one millilitre of VIS (passage 3, T2) was aliquoted on cryogenic vials (Corning Incorporated, Kennebunk, USA) and stored at -196°C. For VLS, 200 µL of T2 of SARS-CoV-2 isolate was added to 800 µL of NucliSENS® Lysis Buffer (bioMérieux) for virus inactivation and conservation of genetic material in cryogenic vials (Corning Incorporated) and stored at -20°C. The delivery of VIS and VLS was made by the Brazilian Mail Company in partnership with Ministry of Science, Technology and Innovations (MCTIC) of Brazil (http://www.mctic.gov.br) in accordance with the Brazilian Health Regulatory Agency (ANVISA) biosafety rules. Upon formal request to acquire VIS and/or VLS, all recipients signed a term of commitment and responsibility for the use of such reagents within the norms stipulated by WHO.13
Araujo D.B., Machado R.R., Amgarten D.E., Malta F.D., de Araujo G.G., Monteiro C.O., Candido E.D., Soares C.P., de Menezes F.G., Pires A.C., Santana R.A., Viana A.D., Dorlass E., Thomazelli L., Ferreira L.C., Botosso V.F., Carvalho C.R., Oliveira D.B., Pinho J.R, & Durigon E.L. (2020). SARS-CoV-2 isolation from the first reported patients in Brazil and establishment of a coordinated task network. Memórias do Instituto Oswaldo Cruz, 115, e200342.
Cerebral cortex tissues were fixed in 2.5% glutaraldehyde for 8 h and rinsed with PBS. Secondary fixation was performed using 1% osmic acid solution for at least 1 h. The tissues underwent a graded dehydration series using ethanol (30%, 50%, and 70% uranyl acetate and 80%, 95%, and 100% uranyl acetate). Following dehydration, the samples were embedded in epoxy resin, cut into ultrathin sections, and stained with uranyl acetate and lead citrate. Images were captured using a transmission electron microscope (TEM) (#JEM1400, Japan).
Zhao J., Chen C., Ge L., Jiang Z., Hu Z, & Yin L. (2024). TAK1 inhibition mitigates intracerebral hemorrhage-induced brain injury through reduction of oxidative stress and neuronal pyroptosis via the NRF2 signaling pathway. Frontiers in Immunology, 15, 1386780.
A drop of the sample was placed on a Formvar-carbon-coated grid for 15 s. The excess droplet was removed using filter paper, and a drop of 1% uranyl acetate was applied for 15 s. After removing the excess uranyl acetate using filter paper, the grid was washed with a drop of DW [93 (link),94 (link)].
Batsukh S., Oh S., Lee J.M., Joo J.H., Son K.H, & Byun K. (2024). Extracellular Vesicles from Ecklonia cava and Phlorotannin Promote Rejuvenation in Aged Skin. Marine Drugs, 22(5), 223.
Mouse spinal cords were fixed in 2.5% glutaraldehyde and 4% PFA in 0.1 M sodium cacodylate buffer at pH 7.4 after deep anesthesia perfusion. Spinal cords were vibratome-sectioned and immersion fixed in the same buffer for 24 h at 4°C. After tissue trimming and washes in 0.1 M sodium cacodylate buffer, postfixation in reduced osmium (2% osmium, 2.5% potassium ferrocyanide in 0.1 M cacodylate buffer) was followed by en bloc uranyl acetate (1% aqueous uranyl acetate) contrasting, graded dehydration in ethanol, and embedding in epon resin (Serva). After ultrathin sectioning, the grids (UC7 Ultramicrotome; Leica) were contrasted by 1% uranyl acetate and lead citrate (Ultrostain; Leica). Semithin sections were contrasted by an equimolar mixture containing 1% methylene blue (Carl Roth GmbH & Co. Kg) in 100 ml sodium tetraborate and 1% (1 g) azure Blue (Carl Roth) in 100 ml water.
Brenner D., Sieverding K., Srinidhi J., Zellner S., Secker C., Yilmaz R., Dyckow J., Amr S., Ponomarenko A., Tunaboylu E., Douahem Y., Schlag J.S., Rodríguez Martínez L., Kislinger G., Niemann C., Nalbach K., Ruf W.P., Uhl J., Hollenbeck J., Schirmer L., Catanese A., Lobsiger C.S., Danzer K.M., Yilmazer-Hanke D., Münch C., Koch P., Freischmidt A., Fetting M., Behrends C., Parlato R, & Weishaupt J.H. (2024). A TBK1 variant causes autophagolysosomal and motoneuron pathology without neuroinflammation in mice. The Journal of Experimental Medicine, 221(5), e20221190.
Five microliters of miR-44438–NCMB was applied to a copper grid with a carbon support membrane. After a 60-s incubation, the excess sample was removed with filter paper and stained with a 2% uranyl acetate solution for 60 s. The excess uranyl acetate solution was removed with filter paper and by air-drying. Electron microscope images were collected using a Tecnai F20 TEM at 200 kV.
Yu Z., Zheng Y., Cai H., Li S., Liu G., Kou W., Yang C., Cao S., Chen L., Liu X., Wan Z., Zhang N., Li X., Cui G., Chang Y., Huang Y., Lv H, & Feng T. (2024). Molecular beacon–based detection of circulating microRNA-containing extracellular vesicle as an α-synucleinopathy biomarker. Science Advances, 10(20), eadl6442.
Samples were fixed with 2.5% glutaraldehyde, 2% paraformaldehyde in 0.1 M sodium cacodylate buffer, postfixed with 1% osmium tetroxide followed by 2% uranyl acetate, dehydrated through a graded series of ethanol and embedded in LX112 resin (LADD Research Industries, Burlington VT). Ultrathin sections were cut on a Leica Ultracut UC7, stained with uranyl acetate followed by lead citrate and viewed on a JEOL 1400 Plus transmission electron microscope at 120kV.
Lagou M.K., Argyris D.G., Vodopyanov S., Gunther-Cummins L., Hardas A., Poutahidis T., Panorias C., DesMarais S., Entenberg C., Carpenter R.S., Guzik H., Nishku X., Churaman J., Maryanovich M., DesMarais V., Macaluso F.P, & Karagiannis G.S. (2024). Morphometric Analysis of the Thymic Epithelial Cell (TEC) Network Using Integrated and Orthogonal Digital Pathology Approaches. bioRxiv.
Sourced in Japan, United States, Germany, United Kingdom, China, France
The Hitachi H-7650 is a transmission electron microscope (TEM) designed for high-resolution imaging of materials. It provides a core function of nanoscale imaging and analysis of a wide range of samples.
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The JEM-1400 is a transmission electron microscope (TEM) produced by JEOL. It is designed to provide high-quality imaging and analysis of a wide range of materials at the nanoscale level. The JEM-1400 offers a maximum accelerating voltage of 120 kV and features advanced optics and detectors to enable detailed examination of samples.
Sourced in Japan, United States, Germany, China, United Kingdom
The HT7700 is a high-resolution transmission electron microscope (TEM) designed for materials analysis and characterization. It provides advanced imaging and analytical capabilities for a wide range of applications in materials science, nanotechnology, and life sciences. The core function of the HT7700 is to enable high-resolution, high-contrast imaging and elemental analysis of nanoscale structures and materials.
Sourced in Japan, United States, Germany, United Kingdom, India
The JEM-1400Plus is a transmission electron microscope (TEM) manufactured by JEOL. It is designed for high-resolution imaging and analysis of a wide range of samples. The JEM-1400Plus provides stable and reliable performance for various applications in materials science, biological research, and other related fields.
Sourced in Germany, Austria, United States, Japan, Switzerland, China, France
The Leica EM UC7 is an ultramicrotome designed for cutting ultrathin sections of samples for transmission electron microscopy (TEM) analysis. It features a precision-engineered cutting mechanism that allows for the preparation of high-quality ultrathin sections with thicknesses ranging from 15 to 500 nanometers.
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Uranyl acetate is a chemical compound used in various laboratory applications. It is a crystalline salt with the formula UO2(CH3COO)2. Uranyl acetate is commonly employed as a staining agent in electron microscopy to enhance the contrast of biological samples during imaging.
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Glutaraldehyde is a chemical compound used as a fixative and disinfectant in various laboratory applications. It serves as a cross-linking agent, primarily used to preserve biological samples for analysis.
Sourced in Japan, United States, Germany, France, United Kingdom
The JEM-1400 is a transmission electron microscope (TEM) manufactured by JEOL. It is designed to produce high-resolution images of small-scale structures and materials. The JEM-1400 uses an electron beam to illuminate and magnify samples, allowing users to study their internal structure and composition at the nanometer scale.
The JEM-1011 is a transmission electron microscope (TEM) manufactured by JEOL. It is designed for high-resolution imaging of various materials and samples. The JEM-1011 provides a maximum accelerating voltage of 100 kV and can achieve a resolution of up to 0.45 nm.
Sourced in Japan, United States, Germany, Spain, China
The JEM-1010 is a transmission electron microscope (TEM) manufactured by JEOL. It is designed for high-resolution imaging of a wide range of samples. The JEM-1010 provides a maximum accelerating voltage of 100 kV and offers a high level of stability and reliability for routine imaging applications.
More about "Uranyl acetate"
Uranyl acetate is a versatile chemical compound with the formula UO2(CH3COO)2.
It is widely used as a staining agent in electron microscopy to enhance the contrast of biological samples, allowing for better visualization and analysis.
This compound is also utilized in the synthesis of other uranium-based compounds and as a precursor for the production of uranium catalysts.
Uranyl acetate has a wide range of applications in various fields, including materials science, nuclear technology, and biochemistry.
Researchers can optimize their uranyl acetate experiments using cutting-edge tools like PubCompare.ai's AI-driven protocol comparison feature.
This innovative tool helps identify the most accurate and reproducible methods from literature, preprints, and patents, enhancing research outcomes.
Some related terms and subtopics include: Uranyl ion (UO2^2+), Acetate ion (CH3COO-), Electron microscopy staining, Transmission electron microscopy (TEM), Scanning electron microscopy (SEM), Biological sample preparation, Nuclear fuel processing, Uranium catalysts, Materials characterization, and Biochemical assays.
Common abbreviations are UA for uranyl acetate and TEM for transmission electron microscopy.
Researchers can leverage the insights from advanced electron microscopes like the H-7650, JEM-1400, HT7700, JEM-1400Plus, and EM UC7 to further optimize their uranyl acetate-based experiments.
Additionally, using complementary chemicals like glutaraldehyde can enhance the effectiveness of uranyl acetate staining in TEM imaging techniques like the JEM-1400, JEM-1011, and JEM-1010.
With the right tools and techniques, scientists can unlock the full potential of uranyl acetate in their research endeavors.
