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753 protocols using em uc7

1

Ultrastructural Analysis of Plant Fronds

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For SEM, the fresh fronds were fixed in 2.5% glutaraldehyde in phosphate-buffered saline (PBS) buffer (1 M, pH 7.4) overnight at 4 °C followed by a stepwise ethanol and tert butanol dehydration. Then samples were dried using a freeze dryer (Hitachi ES-2030). The obtained specimens were examined with a scanning electron microscope (Hitachi S4800) at 30 kV.
For light and transmission electron microscopy (TEM), the samples were washed in PBS buffer after fixing overnight at 4 °C. Then samples were post-fixed with 1% OsO4 in PBS for 2 h at 4 °C following stepwise ethanol and acetone dehydration and infiltration with Spurr’s epoxy resin. The treated samples were embedded and polymerized in Spurr’s epoxy resin at 60 °C for 48 h. Sections for light microscopy were cut using a LEICA EM UC 7 instrument with a glass knife and stained with 1% toluidine blue. The obtained specimens were photographed with an OLYMPUS BX53 camera. Ultra-thin sections (70 nm) for TEM were also cut using a LEICA EM UC 7 instrument and double-stained with 2% uranyl acetate and Sato’s lead citrate. The obtained specimens were examined with a transmission electron microscope (Hitachi-7700) at 120 kV.
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2

Electron Microscopy Sample Preparation

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Cells were fixed with 2.5% glutaraldehyde for 30 min, and post-fixed in 1% buffered osmium tetroxide for 1 h at 20°C. Then, the samples were dehydrated by an increasing ethanol gradient and embedded in epoxy resin. Ultrathin sections (60–80 nm) were sliced using an ultramicrotome (Leica EM UC7; Leica), and observed under a transmission electron microscope (Tecnai G2 20 TWIN; FEI; Thermo Fisher Scientific, Inc.).
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3

Immunolocalization of hpaXm Protein in Transgenic Plants

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Polyclonal antibody (pAb) against hpaXm were customized from Abmart Company (Shanghai, China), by injecting two New Zealand white rabbits respectively with the purified (≥ 3 mg) hpaXm protein [20 ] for three times at 2–3-week intervals. Blood containing the antiserum was collected 2 weeks after the final injection.
Colloidal-gold was preserved in our laboratory. Leaf samples of the tested transgenic lines expressing the full-length and the N-terminal leader peptide deleted mutant of hpaXm were fixed, washed, dehydrated, penetrated, embedded, and polymerized, with the tested vector-expressing transgenic line as control [20 , 44 ]. Then the leaf samples were cut into about 6-nm-thick slices and adsorbed on copper web under the ultramicrotome of Leica-EM UC7 [18 (link), 44 ]. Copper webs containing the leaf samples were moist, closed, and incubated with the diluted hpaXm-pAb twice [18 (link), 44 ]. After being washed and dyed, copper webs were observed under a Hitachi H-7650 electron microscope [18 (link), 44 ]. All tested samples of each group were repeated at least twice.
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4

Ultrastructural Analysis of Cells by Transmission Electron Microscopy

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Approximately 2 × 106 cells were collected by centrifugation at 1,200 rpm for 10 min. The cell pellets were fixed with 2.5 % glutaraldehyde and stored at 4 °C. The cell pellets were washed with 0.1 M sodium cacodylate and further fixed with 4 % osmium tetroxide for 1 h, followed by serial dehydration in ascending concentrations of acetone (from 35 % to 100 %). The samples were infiltrated with a mixture of acetone and resin at the ratio of 1:1 for 1 h, 1:2 for 2 h and 100 % resin overnight as reported [64 (link)]. Finally, the samples were polymerized at 60 °C for 36 h prior sectioning to prepare the 90 nm thick sections using the ultra-microtome Leica EM UC7 (Leica, Germany). The ultrathin sections were stained with lead citrate/uranyl acetate, and analyzed under a transmission electron microscope (Hitachi 7500, Japan).
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5

Ultrastructural Analysis of Mitochrondria in Substantia Nigra

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The rats were euthanized by intraperitoneal injection of 1% pentobarbital (800 mg/kg), and 1 mm3 of substantia nigra tissues was quickly removed and fixed with 2.5% glutaraldehyde at 4°C for 2 h. After fully washing with PBS, the samples were fixed with 1% Russian acid. After fully washing with PBS, the samples were dehydrated with 30, 50, 70, 90, and 100% ethanol for 15 min, and propylene oxide was added to replace the ethanol for 30 min. Then, the samples were embedded with Epon 812 epoxy resin (Ted Pella, CA, USA), placed in the 1:1 mixture of Epon 812 epoxy resin (formula: Epon 812: DDSA: NMA: DMP30 = 27.5:4:20:0.75) and propylene oxide for 2 h, and in the 2:1 mixture for 1 h, then soaked in pure epoxy resin Epon 812 for 2 h, and placed into a 60°C oven for 12 h. The embedded tissues were prepared into 50- to 70-nm-thick ultrathin sections using an ultrathin slicer (Leica EM UC7, Leica, Germany). With 100-mesh copper mesh as the carrier mesh, the sections were stained with uranyl acetate and lead citrate and observed under a TEM (HITACHI H-7650, HITACHI, Japan) to observe the ultrastructural changes in neuronal mitochondria in substantia nigra of rats.
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6

Ultrastructural Analysis of GABA Receptors

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Hippocampal sections obtained from both WT and APP/PS1 mice at 12 months of age were incubated in 10% NGS diluted in TBS.
After several washes in TBS, hippocampal sections were incubated in the primary antibody (anti-GABAB1 at a concentration of 3–5 µg/mL and diluted in TBS containing 1% NGS). After several washes in TBS, the sections were incubated in goat anti-rabbit IgG coupled to 1.4 nm gold (Nanoprobes Inc., Stony Brook, NY, USA). Next, hippocampal sections were postfixed in 1% glutaraldehyde, washed in double-distilled water and silver enhancement of the gold particles (HQ Silver kit, Nanoprobes Inc.). Following treatment with osmium tetraoxide (1% in 0.1 M phosphate buffer), block-staining with uranyl acetate and dehydration in graded series of ethanol, the sections were then flat-embedded on glass slides in Durcupan (Fluka) resin. After polymerization of the resin, regions of interest were cut at 70–90 nm on an ultramicrotome (Leica EM UC7, Leica, Wetzlar, Germany) and collected on pioloform-coated copper grids. Finally, ultrathin sections were stained on drops of 1% aqueous uranyl acetate and Reynolds’s lead citrate. For ultrastructural analyses we used a JEOL-1010 electron microscope.
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7

Ultrastructural Analysis of Induced Cardiomyocytes

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The induced‐hPSC‐derived cardiomyocytes were directly scraped off from the dish and then fixed with 2% glutaraldehyde overnight at 4°C.26 These samples were postfixed with 0.25% osmium/0.25% K4Fe(CN)6, 1% tannic acid, followed with 50 mmol/L uranyl acetate. Then specimens were washed 3 times and dehydrated with a series of ethanol. Finally, the cell samples were embedded in araldite 502 resin (Polysciences Inc, Warrington, PA), and polymerization proceeded at 65°C for several days. The ultrathin sections (≈60 nm) obtained by ultramicrotome (Leica EM UC7; Leica, Wetzlar, Germany)) were mounted in EM‐grids, stained with lead citrate, and then observed by FEI Tecnai G2 Spirit TEM (FEI, Hillsboro, OR).
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8

Quantifying Mitochondrial Damage in Ischemic Cortex

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About 1 mm3 of ischemic cortex tissue was collected and then steeped in 2.5% glutaraldehyde at 4 °C for 2 days. Then the tissue block was fixed with 1% osmic acid, and gradient dehydrated with acetone, then the samples were embedded in propylene oxide and resin. The samples were then sliced with a Leica ultramicrotome (Leica EMUC7, Wetzlar, Germany) and stained with uranyl acetate and lead citrate, which were observed and taken photographs under HT7700 transmission electron microscope (HITACHI, Tokyo, Japan) finally. Five fields were randomly selected in each group and three times were repeated in each group to calculate the proportion of damaged mitochondria, the proportion of mitochondria in each group = the number of damaged mitochondria/the total number of mitochondria.
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9

Electron Microscopy Sample Preparation

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Cells were fixed with 2.5% glutaraldehyde overnight at 4 °C and washed with cold PBS. After postfixing with fixative solution containing 1% osmium tetroxide for 1 h, cells were dehydrated with ascending grades of alcohol. Cells were then infiltrated and embedded in Spurr resin. Ultrathin sections were obtained using Leica EM UC7 (Leica, Wetzetlar, Germany) and stained with uranyl acetate and lead citrate. Cells were observed and photographed under a transmission electron microscope (Hitachi Model H-7650, Tokyo, Japan).
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10

Transmission Electron Microscopy Protocol

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The technique used was described previously (Mei et al., 2018 (link)). The specimen was immersed in 2.5% glutaraldehyde and then postfixed in 2% osmium tetroxide in sodium phosphate buffer for 2 h at 4 ° C. The samples were dehydrated in a graded series of ethanol and propylene oxide. Then, all samples were embedded in araldite. One-micrometer sections were cut with an ultramicrotome (Leica EM UC7; Leica, Nussloch, Germany). After staining with lead citrate and uranyl acetate, the sections were observed using a Hitachi Transmission Electron Microscope (HT7700; Hitachi, Tokyo, Japan).
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