Samples were fixed with 2% v/v glutaraldehyde in 0.05 M sodium phosphate buffer (pH 7.2). Following isolation of suitable specimen blocks, the samples were rinsed three times in 0.15 M sodium phosphate buffer (pH 7.2) and subsequently post-fixed in 1% w/v OsO4 in 0.12 M sodium phosphate buffer (pH 7.2) for 2-hour. The specimens were dehydrated in 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 ultramicrotome (Leica, Vienna, Austria) and collected on copper grids with Formvar supporting membranes (Ted Pella, 01700-F) stained with uranyl acetate (Agar scientific, AGR1260A) and lead citrate (Agar scientific, AGR1210), and subsequently examined with a Philips CM 100 Transmission EM (Philips, Eindhoven, the Netherlands), operated at an accelerating voltage of 80 kV and equipped with an OSIS Veleta digital slow scan 2k x 2k CCD camera. Digital images were recorded with the ITEM software package. Electron microscopy was performed by Klaus Qvortrup, Professor, MD, PhD, University of Copenhagen Faculty of Health and Medical Sciences, CFIM, Copenhagen, Danmark.
Ultracut 7 ultramicrotome
Manufactured by Leica
The Leica Ultracut 7 is an ultramicrotome, a specialized laboratory instrument used for cutting ultra-thin sections of samples for microscopy and analysis. The core function of the Ultracut 7 is to precisely and consistently slice materials into extremely thin specimens, typically ranging from 50 to 500 nanometers in thickness, enabling detailed examination and study of their internal structure and composition.
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Lab products found in correlation
Diamond knife, by Electron Microscopy Sciences (3 mentions)
Ultrascan 1000 camera, by Ametek (2 mentions)
Lead citrate, by Leica (2 mentions)
Diamond knife, by Leica (2 mentions)
Cm120 biotwin transmission electron microscope, by Philips (2 mentions)
Tecnai g2 spirit transmission electron microscope, by Thermo Fisher Scientific (2 mentions)
Agr1260a, by Agar Scientific (1 mentions)
Cm 100 transmission em, by Philips (1 mentions)
Tecnai 20, by Thermo Fisher Scientific (1 mentions)
Vt1000 s vibrating blade microtome, by Leica (1 mentions)
Amw microwave tissue processor, by Leica (1 mentions)
Oneview cmos camera, by Ametek (1 mentions)
Tecnai 20 transmission electron microscope, by Thermo Fisher Scientific (1 mentions)
Uranyl acetate, by Agar Scientific (1 mentions)
1400 plus transmission electron microscope, by JEOL (1 mentions)
Em cpd300, by Leica (1 mentions)
13 protocols using ultracut 7 ultramicrotome
1
Transmission Electron Microscopy Sample Preparation
2
Quantitative Cristae Ultrastructure Analysis
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Cells were washed in PBS, fixed with 2.5% glutardialdehyde and 2% formaldehyde in a buffered solution and postfixed in 1% osmium tetraoxide that had been reduced with 1% potassiumhexacyanoferrate34 (link). The cells were dehydrated in an ascending ethanol series, embedded in TAAB embedding resin, and sectioned on a Leica Ultracut 7 ultramicrotome using a Diatome diamond knife. The sections were counter stained using platinum blue (IBIlabs) and lead citrate (Leica) and visualized in an FEI Tecnai 20 transmission electron microscope. They were photographed at ×27,000 magnification with a Gatan ultrascan 1000 camera. To quantitatively analyze the electron microscopically images a line was drawn in imageJ manually into the cristae starting with the cristae junction proceeding into the cristae volume as far as the image quality was sufficient for image quantification, the curvature of the cristae was not crossing the line or a maximum of 130-nm length was reached. Along the drawn line every 2 nm orthogonally line plots with a width of 10 nm were measured with an ImageJ macro starting from the CJ. Each line plot was halved and the position of the minimal intensity was determined for both sides. The distance of both minimal intensities was set as the distance between opposing sides of the cristae.
3
Ultrastructural Analysis of Blimp1 Mutant Decidua
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For TEM analysis, E6.5 wild-type and Blimp1 mutant decidua were fixed with 4% PFA plus 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer at pH 7.2 for 4 h at RT then overnight at 4 °C. After embedding in 4% agarose in PBS, samples were manually trimmed and thick sections (200 μm) cut using a Leica VT1000 S vibrating blade microtome. The thick sections were processed using a Leica AMW microwave tissue processor, followed by infiltration with TAAB TLV resin over 3 days, and polymerisation for 48 h at 60 °C. Ultrathin (90 nm) sections were cut using a Diatome diamond knife on a Leica Ultracut7 ultramicrotome, post-stained with lead citrate for 5 min, and examined on a Tecnai 12 transmission electron microscope (FEI) equipped with a Gatan OneView CMOS camera.
4
Ultrastructural Visualization of Mitochondrial TMRM
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Cells were incubated in a loading buffer with 81, 13.5, or 1.35 nM TMRM for 30 min, washed with PBS, fixed with 2.5% glutardialdehyde and 2% formaldehyde in a buffered solution, and postfixed in either 2% osmium tetroxide or 1% osmium tetroxide that had been reduced with 1% potassium hexacyanoferrate46 (link). The cells were dehydrated in an ascending ethanol series, embedded in TAAB embedding resin, and sectioned on a Leica Ultracut 7 ultramicrotome using a Diatome diamond knife. The sections were counter-stained using platinum blue (IBIlabs, Boca Raton, FL, USA) and lead citrate (Leica Microsystems, Wetzlar, Germany) and visualized in an FEI Tecnai 20 transmission electron microscope. They were photographed at ×2000 magnification with a Gatan ultrascan 1000 camera.
5
Ultrastructural Analysis of Platelet Flna-Knockout
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Washed platelets from the control and platelet-specific conditional Flna-knockout mice were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH=7.4) for 30 minutes and then washed in 0.1 M phosphate buffer 3 times for 10 minutes. Next, the samples were incubated with 1% osmium tetroxide in 0.1 M phosphate buffer for 1 hour and washed in distilled water 3 times. The samples were stained en bloc with 2.5% aqueous uranyl acetate for 30 minutes and then passed through a series of increasing concentrations of ethanol (30%, 50%, 75%, 90%, and 100%) 3 times. Next, the samples were submerged in a series of ethanol-Epon resin mixtures at different concentrations (25%, 50%, 75%, and 100%) 3 times. The sample was embedded in a BEEM capsule and polymerized in a 60°C oven. Then, 70-nm–thin sections were cut using the Leica Ultracut 7 ultramicrotome and mounted on formvar-coated 200-mesh copper grids. The grids were stained for 10 minutes in 2% uranyl acetate and 5 minutes in Reynold lead citrate. The samples were viewed using the Tecnai Spirit transmission electron microscope, which operated at 120 kV. Images were acquired using a DVC1500M camera (AMT Imaging).
6
Ultrastructural Analysis of Mitochondria-ER Contacts
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Tissue samples (~1µm3) were fixed in 1.25% glutaraldehyde, 4% paraformaldehyde in PBS, 4% sucrose (pH7.2), post-fixed in 1% osmium tetraoxide for an hour and dehydrated in a graded series of ethanol. The samples were incubated in propylene oxide for a final dehydration step followed by a 1:1 epoxy resin:propylene oxide incubation overnight. The samples were incubated in pure epoxy resin overnight (EM912 from EMS Science or Durcupan from Fluka) and cured at 50°C for 48 hours. Semithin (1µm) and ultrathin sections (70 nm) were prepared on a Leica Ultracut 7 ultramicrotome. Semithin sections were stained with Toluidine Blue and areas containing glandular structures were identified and subsequently trimmed for ultrathin sectioning. Ultrathin sections were collected on copper-iridium slot grids (EMS Science), stained with 2% uranyl acetate in water (Agar Scientific) and Reynold’s lead citrate (Fluka).
Electron micrographs were collected at 80kV in a Tecnai 12 and a Jeol 1200 transmission electron microscope. High resolution image analysis (15-20,000 x magnification) was performed to quantify mitochondria-ER contacts in epithelial cells.
Electron micrographs were collected at 80kV in a Tecnai 12 and a Jeol 1200 transmission electron microscope. High resolution image analysis (15-20,000 x magnification) was performed to quantify mitochondria-ER contacts in epithelial cells.
7
Transmission Electron Microscopy of Enucleated Eyes
8
TEM Analysis of Fat Body ncMTOC in Drosophila
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TEM analysis of the MTs at the fat body ncMTOC was performed essentially as described67 (link), except that fat bodies from 30 third instar larve were fixed for 72 hrs in 1 ml Karnovsky’s fixative (EM Sciences Cat#15720). Embedding, staining, sectioning and preparation of grids were performed by the Core Facility at UT Southwestern Medical Center. After three rinses with 0.1 M sodium cacodylate buffer, samples were embedded in 3% agarose and sliced into small blocks (1mm3), rinsed with the same buffer three times and post-fixed with 1% osmium tetroxide and 0.8 % potassium ferricyanide in 0.1 M sodium cacodylate buffer for 1.5 h at room temperature. Samples were rinsed with water and en bloc stained with 4% uranyl acetate in 50% ethanol for 2 h. They were then dehydrated with increasing concentration of ethanol, transitioned into propylene oxide, infiltrated with Embed-812 resin and polymerized in a 60°C oven overnight. Blocks were sectioned with a diamond knife (Diatome) on a Leica Ultracut 7 ultramicrotome (Leica Microsystems) and collected onto copper grids, post stained with 2% aqueous Uranyl acetate and lead citrate. Images were acquired on a Phillips CM120 Biotwin transmission electron microscope at the Florida State University Biological Science Imaging Resource (BSIR).
9
Transmission Electron Microscopy of Brown Adipose Tissue
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For transmission electron microscopy sample collection, we performed cardiac perfusion under ketamine anesthesia with a perfusion buffer (4% paraformaldehyde, 1% glutaraldehyde, and 0.1 M sodium cacodylate, pH 7.4), and we dissected BAT into 1 mm pieces that were then fixed with 2.5% glutaraldehyde and 0.1 M sodium cacodylate, pH 7.4. Further processing of the samples was performed at UTSW Electron Microscopy Core as follows: tissue samples were rinsed in 0.1 M sodium cacodylate buffer and postfixed in 1% osmium tetroxide and 0.8% potassium ferricyanide in 0.1 M sodium cacodylate buffer three times for 3 h at room temperature. After three rinses in water they were stained en bloc with 4% uranyl acetate in 50% ethanol for 2 h. Next, the samples were dehydrated with increasing concentrations of ethanol, transitioned into resin with propylene oxide, infiltrated with Embed-812 resin, and polymerized in a 60 °C oven overnight. Blocks were sectioned with a diamond knife (Diatome) on a Leica Ultracut 7 ultramicrotome (Leica Microsystems) and collected onto copper grids, post stained with 2% aqueous uranyl acetate and lead citrate. Images were acquired on a JEOL 1400 Plus electron microscope and photographed with a BIOSPR16 camera.
10
Transmission Electron Microscopy of C. elegans
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