Phytoplankton were counted fresh using a Beckton Dickinson FACSCalibur flow cytometer with a 488 nm argon laser [55 (link)], with the trigger on chlorophyll red autofluorescence. Viruses were enumerated according to the protocol by Brussaard et al. [56 (link)] with modifications by Mojica et al. [57 (link)]. In short, virus samples were fixed with glutaraldehyde (EM-grade, 0.5% final concentration; Sigma-Aldrich, St. Louis, MO, USA) for 15–30 min at 4 °C, after which the samples were flash-frozen in liquid N2 and stored at −80 °C until analysis. After thawing the samples, they were diluted with Tris-EDTA buffer (1M Tris, 0.5M Na2-EDTA, pH of 8.2), stained with nucleic acid-specific SYBR Green I (0.5 × 10−4 concentration of the commercial stock, Invitrogen Molecular Probes, Eugene, OR, USA) at 80 °C for 10 min in the dark. The trigger was set to green fluorescence. FCS express 5 (De Novo Software, Pasadena, CA, USA) was used for all flow cytometric data analyses, with gating performed on red chlorophyll autofluorescence vs. side scatter for the phytoplankton and SYBR Green fluorescence vs. side scatter for the viruses.
Em grade glutaraldehyde
Manufactured by Merck Group
Sourced in United States, Germany
EM-grade glutaraldehyde is a high-purity fixative used in electron microscopy applications. It serves as a cross-linking agent to preserve the structural integrity of biological samples during specimen preparation for imaging and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Sybr green 1, by Thermo Fisher Scientific (2 mentions)
Paraformaldehyde (pfa), by Merck Group (2 mentions)
Fcs express 5, by De Novo Software (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Tecnai g2 f20, by Thermo Fisher Scientific (1 mentions)
0.1 m phosphate buffer, by Merck Group (1 mentions)
Ultracut uct microtome, by Leica (1 mentions)
Potassium hexacyanoferrate, by Merck Group (1 mentions)
Ultrascan es1000 ccd camera, by Ametek (1 mentions)
Jem 1400 transmission electron microscope, by JEOL (1 mentions)
Osmium tetroxide, by TAAB (1 mentions)
Em uc6, by Leica (1 mentions)
0.1 m cacodylate buffer, by Merck Group (1 mentions)
1400 tem, by JEOL (1 mentions)
Reichert ultracut e ultramicrotome, by Leica (1 mentions)
Transmission electron microscope, by Hitachi (1 mentions)
Hexamethyldisilazane, by Merck Group (1 mentions)
Jsm 6480lv scanning electron microscope, by JEOL (1 mentions)
Axiophot 2, by Zeiss (1 mentions)
Anodisc filter, by Cytiva (1 mentions)
15 protocols using em grade glutaraldehyde
1
Phytoplankton and Virus Enumeration Protocols
2
Visualizing Amyloid-Beta Peptide Interactions
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HRTEM and STEM investigations
were performed using FEI Tecnai (G2 F20) transmission electron microscope
operating at 120 keV. Glutaraldehyde (EM grade) and uranyl acetate
were purchased from Sigma-Aldrich chemicals, Saint Louis, MO, USA.
Carbon-coated copper grids (200 mesh) were purchased from Electron
Microscopy Sciences. An aliquot of Aβ1–42 peptide
(500 μM in 20 mM NaOH) was diluted to 50 μM in 10 mM SPB
(pH 7.4). Stock solutions of tetrapeptides (5 mM in DMSO)20 and 43 (negative control), were diluted to a final
concentration of 250 μM in SPB. Equal volumes (25 μL)
of Aβ1–42 and tetrapeptides20 and 43 were mixed for a concentration ratio of 1:5
(Aβ1–42/test peptides) and incubated at 37
°C for 72 h. After the incubation period, a droplet of the sample
was fixed on the grid by using 0.5% of glutaraldehyde solution. After
washing by ultrapure water, grids were stained negatively by 2% uranyl
acetate. The samples were air-dried and examined. Aβ1–42 alone plus the buffers in similar concentration was considered as
a control. For unbiased classification of fibril morphology and abundance,
each EM grid was investigated at various (>10) positions.
were performed using FEI Tecnai (G2 F20) transmission electron microscope
operating at 120 keV. Glutaraldehyde (EM grade) and uranyl acetate
were purchased from Sigma-Aldrich chemicals, Saint Louis, MO, USA.
Carbon-coated copper grids (200 mesh) were purchased from Electron
Microscopy Sciences. An aliquot of Aβ1–42 peptide
(500 μM in 20 mM NaOH) was diluted to 50 μM in 10 mM SPB
(pH 7.4). Stock solutions of tetrapeptides (5 mM in DMSO)
concentration of 250 μM in SPB. Equal volumes (25 μL)
of Aβ1–42 and tetrapeptides
(Aβ1–42/test peptides) and incubated at 37
°C for 72 h. After the incubation period, a droplet of the sample
was fixed on the grid by using 0.5% of glutaraldehyde solution. After
washing by ultrapure water, grids were stained negatively by 2% uranyl
acetate. The samples were air-dried and examined. Aβ1–42 alone plus the buffers in similar concentration was considered as
a control. For unbiased classification of fibril morphology and abundance,
each EM grid was investigated at various (>10) positions.
3
Electron Microscopy Sample Preparation
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Living cells were fixed 24 h after treatments with 2% (w/v) paraformaldehyde (Sigma-Aldrich) and 3% (v/v) glutaraldehyde (EM grade, Merck, Darmstadt, Germany) in 0.1 M phosphate buffer (Sigma-Aldrich), following the protocol described elsewhere [14 (link)]. Briefly, the samples were post-fixed with OsO4 1%, washed several times with distilled H2O, and dehydrated in acetone, embedded in Eponate resin at 60°C for 48 h, and sectioned with an ultramicrotome. Finally, ultrathin sections placed in copper grids were contrasted with uranyl acetate and Reynolds lead citrate solutions, and examined using a Jeol 1400 (Jeol LTD, Japan) TEM equipped with a CCD GATAN ES1000w Erlangshen camera.
4
Ultrastructural Analysis of Motor Neurons
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Perfused spinal cords in a fixative solution of 2.5% (v/v) glutaraldehyde (EM grade, Merck) and 2% (w/v) PFA in 0.1 M PBS, pH 7.4 were incubated for 2 h at room temperature on a rocking platform. Samples were fixed in 1% (w/v) PFA and subsequently post-fixed with 1% (w/v) osmium tetroxide (TAAB Laboratories) containing 0.08% (w/v) potassium hexacyanoferrate (Sigma-Aldrich) in PBS, pH 7.4 for 2 h at 4 ºC. After four washes with deionized water, we dehydrated samples with sequential washes of acetone. We embedded samples in EPON resin and polymerized at 60 ºC for 48 h and then cut into semi- thin sections (1 µm) with a Leica ultracut UCT microtome (Leica Microsystems). Sections were stained with 1% (w/v) aqueous toluidine blue solution and examined with a light microscope to identify the ventral horn area. Ultra-thin sections (70 nm) were then cut with a diamond knife, placed on coated grids, and contrasted with conventional uranyl acetate and Reynolds lead citrate solutions. Finally, we observed the sections with a JEM-1400 transmission electron microscope (Jeol) equipped with a Gatan Ultrascan ES1000CCD camera. We analyzed three to four MNs per animal (n=4 per group).
5
Cellular Uptake of TiO2 Nanoparticles
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Cellular uptake of TiO2 NPs was measured by transmission electron microscopy (TEM). V79-4 cells were grown on 6-well plates at a density of 1.75 × 105 cells/well. Cells were exposed to TiO2 NPs dispersed according to DP1 and DP2 (3, 10, 30 μg/cm2) for 24 h. At the end of the exposure time, cells were centrifuged, fixed in 2.5% (v/v) glutaraldehyde (EM grade, Merck, Darmstadt, Germany) and 2% (w/v) paraformaldehyde (EMS, Hatfield, PA, USA) in 0.1 M cacodylate buffer at pH 7.4 (PB, Sigma-Aldrich, Steinheim, Germany), and processed following conventional procedures, as previously described [23 (link)]. Samples were first post-fixed with osmium tetroxide, dehydrated in acetone, later embedded in Epon, and finally polymerized at 60 °C, and cut with an ultramicrotome Leica EM UC6 using a diamond knife and mounted on copper grids. Before image acquisition, sections were stained using uranyl acetate and Reynolds lead-citrate solutions. All images were examined using a JEOL 1400 (JEOL LTC, Tokyo, Japan) TEM at 120 kV equipped with a CCD GATAN ES1000W Erlangshen camera.
6
TEM Analysis of PSNPL Uptake Effects
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TEM was used to determine potential changes in the cellular structure induced by PSNPLs uptake. After a 24, 48, or 72 h exposure to 25 μg/mL PSNPLs, cells were washed with PBS 1X, trypsinized, pelleted, and fixed in 2.5 % (v/v) glutaraldehyde (EM grade, Merck, Darmstadt, Germany) and 2 % (w/v) paraformaldehyde (EMS, Hatfield, USA), in 0.1 M cacodylate buffer (PB, Sigma-Aldrich, Germany) at pH of 7.4. Then, sampling processing was carried out following conventional procedures, as previously reported (Annangi et al., 2015) (link). A Jeol 1400 TEM (Jeol LTD, Tokyo, Japan) equipped with a CCD GATAN ES1000W Erlangshen camera was used to take representative cell images.
7
Histopathological Analysis of Aged Mouse Hearts
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The hearts were collected at 8 months of age, fixed in 10% buffered formalin, and embedded in paraffin. Serial 5-μm heart sections from each group were stained with hematoxylin and eosin or Masson's trichrome [17] . For electron microscopy, the left ventricles were dissected from hearts, and fixed in 2% EM-grade glutaraldehyde (Sigma), 2% PFA in 0.2 M sodium cacodylate (pH 7.4; Sigma) overnight at 4 °C, and post-fixed in 1% osmium tetroxide (EM Sciences) in 0.2 M sodium cacodylate (pH 7.4) for 2 h at 4 °C. Tissue was treated with propylene oxide and resin embedded (EMBED 812 kit, EM Sciences). The osmicated samples were then dehydrated through a series of graded ethanol solutions and then infiltrated with propylene oxide (EM Sciences). Subsequently, the tissues were infiltrated with Durcupan ANC Fluka Araldite (Sigma Chemical) and embedded in the same resin. After polymerization, 80-nm sections were cut using a Reichert Ultracut E Ultramicrotome (Leica Microsystems, Bannockburn, IL), and the grids were subsequently stained with uranyl acetate and lead citrate. The sections were imaged using a Hitachi transmission electron microscope equipped for digital image acquisition.
8
SEM Imaging of Mouse Blood Cells
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Scanning electron microscopy (SEM) was performed as described previously (Huang et al. 2016 (link)). Mouse blood was fixed overnight in 3% EM-grade glutaraldehyde (Sigma-Aldrich) at 4° immediately upon collection. The samples were washed with MT-PBS 3 times, 10 min each time. The cells were then adhered to the coverslips with 0.1% polyethylenimine (PEI) for 10 min, before washing with MT-PBS. The cells were then dried serially using 30, 50, 70, 80, 90, 100, and 100% ethanol, for 10 min each. The cells were then soaked in 1:1 ethanol: hexamethyldisilazane solution for 10 min, followed by two washes with 100% hexamethyldisilazane (Sigma-Aldrich), each for 10 min. The coverslips were then air dried overnight, coated with gold, and then examined under a JEOL JSM-6480LV scanning electron microscope.
9
Quantifying Viral Abundance in Water
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Freshly collected water samples were immediately transferred into 4 mL cryovials, fixed for 15–30 min in 0.5% EM-grade glutaraldehyde (Sigma, USA) and subsequently flash frozen in liquid nitrogen. For long-term storage, samples were transferred to −80 °C until preparation. For this purpose, samples were thawed in a 37 °C water bath and then homogenized with a vortex mixer, before 1 mL was transferred into 1.5 mL Eppendorf vials (for highly turbid samples the volume was reduced to 250 μL and raised up to 1 mL with MQ water previously filtered through a 0.02 μm pore size filter). The filtration unit was mounted with a pre-wetted 0.45 μm nitrocellulose support filter and a 0.02 μm Anodisc filter (Whatman, UK) was carefully placed on top of it. The filtration funnel was clamped onto the soaked filters, the sample was vortexed for 3–5 s, immediately before dripping it slowly in circles onto the Anodisc to guarantee an even sample distribution. Using a hand-operated vacuum pump (≤13 kPa), the samples were slowly filtered onto the Anodisc filter, which was then carefully removed while vacuum was still on and after it became dry, it was stained with SYBR Gold. Stained virus-like particles (VLP) were counted with an epifluorescence microscope (Axiophot 2, Zeiss, Germany) at 1250–2500x magnification.
10
Methanotroph Characterization Protocol
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