Control HCT116 cells, and those treated with sulindac sulfide were analyzed for ER morphology by TEM as previously described [50 (link)]. For immunogold TEM, cells were fixed in cold 2% paraformaldehyde in 0.01M PBS (pH 7.4), rinsed in PBS, dehydrated through a graded series of ethanol, and infiltrated with and embedded in LR White resin (Electron Microscopy Sciences). Semi-thin (300 nm) sections were obtained on a Leica Ultracut 7, stained with 0.5% Toluidine Blue in 1% sodium borate, and examined under the light microscope to determine specific areas. Ultrathin sections (65 nm) were picked up on 100 mesh nickel grids, labeled with 1:100 dilution of rabbit anti-BiP (Abcam) overnight at 4°C, and then labeled with a 6 nm goat anti-rabbit colloidal gold conjugated secondary at a dilution of 1:10 at room temperature for 1 hr. After rinsing in PBS and dH2O, the sections were counterstained with 2% uranyl acetate and examined on JEOL 1011 transmission electron microscope with a side mount AMT 2k digital camera (Advanced Microscopy Techniques).
Ultracut 7
Manufactured by Leica
Sourced in Austria
The Leica Ultracut 7 is a high-performance ultramicrotome designed for the preparation of ultrathin sections for electron microscopy. It features precise and reliable sectioning capabilities, ensuring consistent and uniform sample preparation.
Automatically generated - may contain errors
Lab products found in correlation
Cm 100 transmission em, by Philips (4 mentions)
Amt 2k digital camera, by Advanced Microscopy Techniques (2 mentions)
Rabbit anti bip, by Abcam (2 mentions)
Lr white resin, by Electron Microscopy Sciences (2 mentions)
Oneview cmos camera, by Ametek (1 mentions)
Tecnai 12, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Glutaraldehyde, by Polysciences (1 mentions)
Jem 2100, by JEOL (1 mentions)
Formvar coated copper grid, by Electron Microscopy Sciences (1 mentions)
Tecnai g2 spirit biotwin, by Thermo Fisher Scientific (1 mentions)
Tem 2100, by JEOL (1 mentions)
14 protocols using ultracut 7
1
Ultrastructural Analysis of ER Morphology
2
Ultrastructural Analysis of KCa3.1 Silencing in TGF-β1 Treated Cells
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HK2 cells grown on glass coverslips were transfected with KCa3.1 or scrambled siRNA overnight and then incubated with TGF-β1 for 48 hours as described above. The samples were prepared for transmission electron microscopic analysis as previously reported48 (link). Briefly, cells were washed with pre-warmed PBS twice, and then fixed in 2% glutaraldehyde in PBS for 60 minutes at room temperature. Fixed cells were washed with PBS 3 times then postfixed with 1% in osmium tetroxide in PBS for 1hr. After rinsing 3 times with distilled water, the samples were further stained with 1% tannic acid for 1 hour. Finally, the cells were infiltrated and double-embedded in Epon. Sections of 70 nm were generated with an ultramicrotome (Ultracut 7, Leica) and post-stained with 2% aqueous uranyl acetate and Reynold’s lead citrate for 10 min each. The specimens were examined with the JEOL 2100TEM at 200 kV.
3
Transmission Electron Microscopy of Oxacillin-Treated Cells
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Strains were grown in TSB at 37°C as specified above, but with an initial OD600 of 0.02, and at an OD600 of 0.2 the culture was divided in two: one without oxacillin and one supplemented with oxacillin at 1.25 μg ml−1. The cultures were incubated at 37°C for 20 min. The cells were collected by centrifugation (8,000 × g; 5 min) and suspended in fixation solution as described above, followed by incubation overnight at 4°C. The fixed cells were further embedded in agarose, rinsed three times in 0.15 M sodium phosphate buffer (pH 7.2), and subsequently postfixed in 1% OsO4 with 0.05 M K3Fe(CN)6 in 0.12 M sodium phosphate buffer (pH 7.2) for 2 h. The specimens were dehydrated in a graded series of ethanol, transferred to propylene oxide, and embedded in Epon according to standard procedures. Sections, approximately 60 nm thick, were cut with an Ultracut 7 (Leica, Wienna, Austria) and collected on copper grids with Formvar supporting membranes, stained with uranyl acetate and lead citrate. Specimens examined with a Philips CM 100 transmission EM (Philips, Eindhoven, The Netherlands), operated at an accelerating voltage of 80 kV. Digital images were recorded with an Olympus Veleta digital slow scan 2.048 × 2.048 charge-coupled device camera and the ITEM software package.
All SEM and TEM processing and microscopy of fixed cells were performed at the CFIM.
All SEM and TEM processing and microscopy of fixed cells were performed at the CFIM.
4
Transmission Electron Microscopy Sample Prep
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Pellets of bacteria were fixed with 2% (vol/vol) glutaraldehyde in 0.05 M sodium phosphate buffer (pH 7.2). The pellets were embedded in agarose, rinsed three times in 0.15 M sodium phosphate buffer (pH 7.2), and subsequently postfixed in 1% (wt/vol) OsO4 with 0.05 M K3Fe(CN)6 in 0.12 M sodium phosphate buffer (pH 7.2) for 2 hours. The specimens were dehydrated in a graded series of ethanol, transferred to propylene oxide, and embedded in Epon according to standard procedures. Sections, approximately 60-nm thick, were cut with a Ultracut 7 (Leica, Wienna, Austria) and collected on copper grids with Formvar supporting membranes, stained with uranyl acetate and lead citrate, and subsequently examined with a Philips CM 100 Transmission EM (Philips, Eindhoven, The Netherlands), operated at an accelerating voltage of 80 kV. Digital images were recorded with an OSIS Veleta digital slow scan 2k × 2k CCD camera and the ITEM software package.
5
Transmission Electron Microscopy Sample Prep
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Pellets of bacteria were fixed with 2% (vol/vol) glutaraldehyde in 0.05 M sodium phosphate buffer (pH 7.2). The pellets were embedded in agarose, rinsed three times in 0.15 M sodium phosphate buffer (pH 7.2), and subsequently postfixed in 1% (wt/vol) OsO4 with 0.05 M K3Fe(CN)6 in 0.12 M sodium phosphate buffer (pH 7.2) for 2 hours. The specimens were dehydrated in a graded series of ethanol, transferred to propylene oxide, and embedded in Epon according to standard procedures. Sections, approximately 60-nm thick, were cut with a Ultracut 7 (Leica, Wienna, Austria) and collected on copper grids with Formvar supporting membranes, stained with uranyl acetate and lead citrate, and subsequently examined with a Philips CM 100 Transmission EM (Philips, Eindhoven, The Netherlands), operated at an accelerating voltage of 80 kV. Digital images were recorded with an OSIS Veleta digital slow scan 2k × 2k CCD camera and the ITEM software package.
6
Ultrastructural Analysis of ER Morphology
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Control HCT116 cells, and those treated with sulindac sulfide were analyzed for ER morphology by TEM as previously described [50 (link)]. For immunogold TEM, cells were fixed in cold 2% paraformaldehyde in 0.01M PBS (pH 7.4), rinsed in PBS, dehydrated through a graded series of ethanol, and infiltrated with and embedded in LR White resin (Electron Microscopy Sciences). Semi-thin (300 nm) sections were obtained on a Leica Ultracut 7, stained with 0.5% Toluidine Blue in 1% sodium borate, and examined under the light microscope to determine specific areas. Ultrathin sections (65 nm) were picked up on 100 mesh nickel grids, labeled with 1:100 dilution of rabbit anti-BiP (Abcam) overnight at 4°C, and then labeled with a 6 nm goat anti-rabbit colloidal gold conjugated secondary at a dilution of 1:10 at room temperature for 1 hr. After rinsing in PBS and dH2O, the sections were counterstained with 2% uranyl acetate and examined on JEOL 1011 transmission electron microscope with a side mount AMT 2k digital camera (Advanced Microscopy Techniques).
7
Ultrastructural Analysis of Mature and Developing Sperm
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To visualise the ultrastructure of mature and developing sperm, transmission electron microscopy was utilised77 (link). Epididymis and testis from adult mice were dissected as previously described78 (link), cut into 1–2 mm3 cubes and fixed for at least 24 h at 4 °C in 2.5% glutaraldehyde (Polysciences) and 2% PFA (Sigma) in 0.1 M 1,4 Piperazine bis(2-ethanosulfonic acid) (PIPES) buffer (pH = 7.2, Sigma-Aldrich). Samples were washed in 0.1 M PIPES buffer with 50 mM glycine before a second fixation step with 1% osmium tetroxide in 0.1 M PIPES for 1 h at 4 °C and a tertiary fixation in 2% uranyl acetate for 2 h at 4 °C. Tissue was then dehydrated in gradient steps of ethanol and embedded in epoxy-resin. Ultrathin sections were cut in an ultramicrotome (Ultracut 7, Leica) and imaged using a Tecnai 12 (FEI) Transmission Electron Microscope operated at 20 kV with a OneView CMOS camera (Gatan). Images were adjusted for brightness/contrast with GIMP Image Editor (GIMP Development Team).
8
Ultrastructural Analysis of Extracellular Vesicles
9
Transmission Electron Microscopic Analysis of Mitochondrial Morphology
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The cell samples were prepared for transmission electron microscopic analysis as previously reported (Huang et al., 2014a (link)). Briefly, after washing with pre-warmed PBS, the cells were next fixed in 2% glutaraldehyde for 1 h. Subsequently, the fixed cells were postfixed with 1% osmium tetroxide for 1 h after briefly washing with PBS. The samples were rinsed in distilled water, stained with 1% tannic acid, dehydrated in a gradient of ethanol, and embedded in Epon. Sections of 70 nm were generated with an ultramicrotome (Ultracut 7, Leica) and post-stained with 2% aqueous uranyl acetate and Reynold’s lead citrate for 10 min each. The specimens were examined under a transmission electron microscope operating at 200 kV (JEM-2100, JEOL, Japan). Mitochondrial Feret’s diameter (maximum and minimum), the distance between two parallel tangential lines within the selected mitochondrion, was determined using Image J (Demeter-Haludka et al., 2018 (link); Lomash et al., 2019 (link)).
10
Ultrastructural Analysis of Cell Fractions
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Pellets with Hela cells, liver and kidney homogenate constituting the starting materials and pellets formed by centrifugation after each step in the fractionation protocol were fixed with 2% v/v glutaraldehyde in 0.05 M sodium phosphate buffer (pH 7.2). The pellets were then embedded in agarose, rinsed three times in 0.15 M sodium phosphate buffer (pH 7.2), and subsequently post-fixed in 1% w/v OsO4 with 0.05 M K3Fe(CN)6 in 0.12 M cacodylate buffer (pH 7.2) for 2 hrs. The specimens were then dehydrated in graded series of ethanol, transferred to propylene oxide and embedded in epon (812 Resin kit, TAAB Laboratories Equipment Ltd, Aldermaston, UK) according to standard procedures.
Sections, approximately 60 nm thick, were cut with an Ultracut 7 (Leica, Vienna, Austria) and collected on formvar coated copper grids (Electron Microscopy Sciences, Hatfield, PA), stained with 0.5 % w/v uranyl acetate and 3 % w/v lead citrate. Subsequently, they were examined with a Philips CM 100 Transmission EM (Philips, Eindhoven, The Netherlands), operated at an accelerating voltage of 80 kV. Digital images were recorded with an OSIS Veleta digital slow scan 2k x 2k CCD camera (Olympus, Münster, Germany) and the iTEM software package.
Sections, approximately 60 nm thick, were cut with an Ultracut 7 (Leica, Vienna, Austria) and collected on formvar coated copper grids (Electron Microscopy Sciences, Hatfield, PA), stained with 0.5 % w/v uranyl acetate and 3 % w/v lead citrate. Subsequently, they were examined with a Philips CM 100 Transmission EM (Philips, Eindhoven, The Netherlands), operated at an accelerating voltage of 80 kV. Digital images were recorded with an OSIS Veleta digital slow scan 2k x 2k CCD camera (Olympus, Münster, Germany) and the iTEM software package.
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