For chemical fixation of Arabidopsis roots, 5-mm root tips of 5-day-old Arabidopsis expressing UBQ10::GFP:Exo84c and VAP27-3p::VAP27-3:mCh following 1 μM Conc A treatment were collected and checked with confocal microscopy, then they were prefixed as in 2.5% glutaraldehyde (v/v in 0.1 M phosphate buffer, pH 7.2) for 2 h. Then the samples were rinsed with 0.1 M phosphate buffer for three times, 15 min each. The samples were post-fixed in 1% OsO4 for another 2 h, and rinsed with 0.1 M phosphate buffer for three times as before. All the samples were dehydrated using an acetone gradient series of 30, 50, 70 and 90%, with a 20 min exposure to each acetone gradient and ending with the dehydration step in 100% acetone for three times (15 min each). The samples were then infiltrated in a graded scale of 3:1, 1:1, 1:3 (v/v) acetone/SPI-PON 812 resin and 100% SPI-PON 812 resin (SPI Supplies, West Chester, PA, USA) at the last step, each step was performed for 12 h. Samples were finally embedded in SPI-PON 812 resin and polymerized at 60 °C for 48 h. For transmission electron microscopy (TEM) analysis, the embedded samples were sectioned on an EM UC7 ultramicrotome (Leica, Germany). Ultrathin sections (80 nm thick) were stained with 2 % uranyl acetate and lead citrate and viewed using a TEM (Hitachi H-7650, Japan) at 80 kV.
Spi pon 812 resin
Manufactured by SPI Supplies
Sourced in United States
The SPI-PON 812 resin is a laboratory equipment product. It is a type of epoxy resin formulation designed for use in various applications. The core function of the SPI-PON 812 resin is to provide a stable and durable material for specific experimental or analytical purposes.
Automatically generated - may contain errors
Lab products found in correlation
Uranyl acetate, by SPI Supplies (4 mentions)
H 7650, by Hitachi (3 mentions)
Lead citrate, by SPI Supplies (2 mentions)
Spi pon 812 resin, by Leica (2 mentions)
Potassium ferrocyanide, by Merck Group (2 mentions)
Em uc7, by Leica (2 mentions)
Em uc7 ultramicrotome, by Leica (2 mentions)
P35g 1.5 14 c grid, by MatTek (1 mentions)
Jem 1400, by JEOL (1 mentions)
Lr white, by SPI Supplies (1 mentions)
Tecnai g2 20 twin electron microscope, by Thermo Fisher Scientific (1 mentions)
Em hpm100, by Leica (1 mentions)
Lead citrate, by Thermo Fisher Scientific (1 mentions)
Uranyl acetate, by Thermo Fisher Scientific (1 mentions)
Osmium tetroxide, by Ted Pella (1 mentions)
12 protocols using spi pon 812 resin
1
Chemical Fixation of Arabidopsis Roots
2
Ultrastructural Analysis of ER-Mitochondria Associations
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All samples were prefixed in 2.5% glutaraldehyde (v/v in 0.1 M phosphate buffer, pH 7.2) for 2 h, and then rinsed 3 times with 0.1 M phosphate buffer (pH 7.2). They were post-fixed in 1% OsO4 for 2 h, followed by three 15 min rinses with phosphate buffer. Afterwards, the samples were dehydrated through an acetone series (30, 50, 70, 90, 100, 100, and 100%) (v/v in dd H2O) at room temperature, samples were incubated for 20 min at each concentration. Then the samples were infiltrated in a graded scale of 3:1, 1:1, 1:3 (v/v) acetone/SPI-PON 812 resin and, as the last step, in 100% (v/v) SPI-PON 812 resin (SPI Supplies, West Chester), for 12 h per step. Samples were embedded in SPI-PON 812 resin and polymerized at 60 °C for 48 h. Ultrathin sections (80 nm) were prepared using an EM UC7 Ultracut ultramicrotome (Leica, UC7). Sections were observed and photographed using a transmission electron microscope (Hitachi H-7650) at an accelerating voltage of 80.0 kV. For ER-mitochondria quantification, the shortest distance between mitochondria and associated ER membrane was measured and at least 20 mitochondria from three independent biological samples were analysed.
3
Ultrastructural Analysis of Transfected Cells
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Transfected ACCs were cultured on 35‐mm glass‐bottomed dishes with a positive marker (MatTek Corp., P35G‐1.5‐14‐C‐GRID). After 3 days of culture for SgII‐KD and 1 day for SgIII‐OE, successfully transfected cells were spotted by EGFP/mCherry under a fluorescence microscope. After selection, cells were fixed with 2% glutaraldehyde plus 2% PFA for 1 h at room temperature and post‐fixed by osmium tetroxide with 1% aqueous uranyl acetate. The cells were then dehydrated through a graded alcohol series and embedded in SPI‐Pon 812 resin (SPI Supplies, PA). Subsequently, sections were cut at 70 nm on an ultramicrotome (Leica Microsystem, UC7) and a 120‐kV transmission electron microscope (JEOL, JEM‐1400) was used in all EM imaging.
4
Immuno-electron Microscopic Analysis of Rickettsia Infection
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For immuno-electron microscopic analysis [26] (link), R. rickettsia-infected Vero cells were treated with fixing agent (4% paraformaldehyde, 0.5% glutaraldehyde, 0.3% picric acid, 0.1M sodium cacodylate, pH 7.4) for 4 h on ice. Then, the cells were sequentially dehydrated with 50%, 70%, 85%, and 95% alcohol and successively permeated in a mixture of LR White (Spi Supplies, West Chester, PA) and alcohol or LR White alone according to the standard method. The samples were embedded in Spi-Pon 812 resin (Spi Supplies, West Chester, PA) and transferred to 200 mesh nickel gird (BeiJingZhongXingBaiRui Technology Co., ltd, Beijing, China). The grids were then incubated with each immune serum (1∶10 dilution) for 2 h. After washing with blocking buffer, the girds were incubated with a goat anti-mouse IgG labeled with 10 nm colloidal gold particles (Aurion, EMS) (1∶20 dilution) for 2 h at room temperature. Following washing, the grids were fixed in 1% glutaraldehyde for 10 min, washed, and stained with uranyl acetate (Spi Supplies, West Chester, PA) and lead citrate (Spi Supplies, West Chester, PA). Finally, the girds were examined using a transmission electron microscopy (TEM) at 80 kV (H-7650, Hitachi Chemical co., Ltd, Japan).
5
Ultrastructural Analysis of Neutrophils
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After being incubated for 2–4 h, neutrophils were pelleted and fixed with 4% glutaraldehyde in 0.1 M PBS overnight at 4°C. Subsequently, Postfixed in 1% osmium tetroxide was followed by dehydration with graded series of ethanol, infiltration, and embedding in SPI-PON 812 resin (SPI Supplies, West Chester, PA, USA). Ultrathin sections with a thickness of 65 nm were cut using a microtome Leica EM UC7 (Leica Microsystems Company, Wetzlar, Hessen, Germany) and poststained with 2% uranyl acetate for 10 min and 0.3% lead citrate for 10 min. The ultrathin sections were observed using a transmission electron microscope (H-7650, Kyoto, Japan).
6
Transmission Electron Microscopy of Embryo Sections
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The general procedures of preparing conventional samples for TEM, described previously, were followed with minor modifications.26 (link) Briefly, middle areas of embryo sections (1 mm3) (StageI,StageII,StageIII,Stage IVin different GA3 treatment) were cut from seedlings (1-week old) and fixed immediately in 2.5% glutaraldehyde in PBS(Phosphate Buffer Saline) overnight at 4°C. The tissues were rinsed three times using PBS, post-fixed in 1% osmium tetroxide for 4 h, rinsed three times using PBS again, dehydrated in a graded acetone series, and embedded in SPI-PON 812 resin (SPI Supplies). For TEM, the ultrathin sections were contrasted with uranyl acetate and lead citrate and were observed directly under the TEM (HT7700, Japan) at 120 kV.
7
Ultrastructural Analysis of Cells
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Cells were washed twice with 0.1 mol/L sodium cacodylate buffer (pH = 7.4) followed by fixation with 2.5% glutaraldehyde in the same buffer for 2 h, and then post-fixed with 1% OsO4 for 1 h at room temperature. After rinsing several times in cacodylate buffer and distilled water, the cells were incubated in 0.1% tannic acid (in cacodylate buffer) for 30 min, and stained in 1% uranyl acetate for 1 h. They were washed again in distilled water and dehydrated in a graded ethanol series and embedded in SPI-Pon 812 resin (SPI Supplies, PA, USA). Ultrathin (70 nm) sections were cut using an ultramicrotome (UC7, Leica Microsystem), and collected on copper grids with a single slot, stained with uranyl acetate and lead citrate. Then the sections were observed under a Tecnai G2 20 TWIN electron microscope at 120 kV and photographed with an Eagle (4k×4k) digital camera (FEI, Oregon, USA).
8
Ultrastructural Imaging of Adult Worms
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Adult worms were loaded onto a 50-μm–thick aluminum specimen carrier and rapidly frozen with a Leica EM HPM100 high-pressure freezing system(Leica Microsystems GmbH, Wetzlar, Germany). After freezing, carriers were transferred into 2 ml microcentrifuge tube containing 1 ml acetone solution of 1% osmium tetroxide and 0.1% uranyl acetate (Ted Pella, Inc., Redding, CA, USA) under liquid nitrogen. The tubes were then placed in Leica EM AFS2 machine(Leica Microsystems GmbH, Wetzlar, Germany) and processed using standard FSF program: −90°C for 48 h, −60°C for 24 h, −30°C for 18 h, and finally to 4°C. Freeze-substituted fixed specimens were washed 3 times with pure acetone and infiltrated with SPI-PON 812 resin(SPI Supplies, West Chester, PA, USA). The specimens were subsequently embedded in a flat mold and polymerized at 60°C; 90 nm sections were obtained with a Leica EM UC7 Ultramicrotome (Leica Microsystems GmbH, Wetzlar, Germany) and picked on 200 mesh copper grids. Sections were poststained with 2% uranyl acetate and Reynold’s lead citrate to enhance contrast and imaged on an FEI Tecnai G2 Spirit (120 kV) electron microscope (FEI Company, Hillsboro, OR, USA).
9
Immunoelectron Microscopy of R. heilongjiangensis
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Vero cells were cultured in Dulbecco's modification of Eagle's medium (DMEM) (Hyclone, Beijing, China) that was supplemented with 5% fetal bovine serum (FBS; Hyclone, San Jose, CA) and infected with R. heilongjiangensis [21 (link)]. For immunoelectron microscopy [2 , 22 (link)], the R. heilongjiangensis-infected Vero cells were collected after 48 h of culture, and then fixed, dehydrated, and embedded in Spi-Pon 812 resin (Spi Supplies, West Chester, PA, USA) and transferred to a 200-mesh nickel grid (BeiJingZhongXingBaiRui Technology Co., Ltd., Beijing, China) as previously described [2 ]. The grids were then incubated with RpsB or TrxA immunized serum (1:10 dilution) for 2 h. After washing, the grids were incubated with goat anti-mouse IgG that was labeled with 10 nm colloidal gold particles (Aurion, EMS) (1:20 dilution) for 2 h, following which, they were washed, fixed in 1% glutaraldehyde for 10 min, stained with uranyl acetate (Spi Supplies, West Chester, PA) and lead citrate (Spi Supplies, West Chester, PA), and examined by transmission electron microscopy (TEM) at 80 kV (H-7650, Hitachi Chemical co., Ltd, Japan) [2 ].
10
Electron Microscopy Sample Preparation
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Samples were fixed in a refrigerator overnight at 4 °C. Fixative solution was discarded and neurons were stained with 1% OsO 4 (Ted Pella) and 1.5% potassium ferrocyanide (Sigma-Aldrich) in 0.1 M cacodylate/HCl buffer for 40 min after three washes with 0.1 M cacodylate/HCl buffer. The samples were post-fixed with 1% OsO 4 in 0.1 M cacodylate/HCl buffer for 40 min followed by three washes with 0.1 M cacodylate/HCl buffer. The samples were then stained with 2% uranyl acetate (SPI supplies) in water for 1 h. After staining, samples were dehydrated in a graded series of ethanol and infiltrated by Spi-Pon 812 resin (SPI supplies). A Spi-Pon 812 resin-filled tube was placed on top of the PDMS substrate before the resin was polymerized in an oven at 60 °C for 48 h. The PDMS was peeled off from the resin block after resin polymerization. Neurons were left in the resin, and patterns of the PDMS were also transferred to the resin block (Fig. 1b and2g andh). The resin block surface was trimmed using a razor blade so that only one square imaged by FLM was at the apex of the resin block. 200 nm sections were acquired in an ultramicrotome (Leica EM UC7). All of the serial sections containing patterns and neuropils were collected on EM grids. The sections were contrasted with uranyl acetate and lead citrate (Alfa Aesar) prior to EM imaging.
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