Conidial suspension of B. cinerea (106 conidia mL−1) was mixed with PDB amended with PB, CCh, and SSi at 0.2% was incubated at 23 ± 1 °C for 8 h. PDB mixed with conidium suspension was served as a control. Samples were centrifuged at 6,000 rpm for 10 min and pellet was fixed in 3% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) at 4 °C. Samples were washed with 0.1 M sodium cacodylate buffer (pH 7.2) for 15 min three times. Subsequently, the samples were post-fixed in 1% osmium tetroxide in 1% cacodylate buffer, embedded in Araldite resin (Electron microscopy Sciences, Hatfield, PA, USA). Stained 0.5 µm sections of TEM blocks were used to select appropriate sections. Ultrathin sections (60 nm) were obtained by using a ultramicrotome (Leica Ultracut UCT, Solms, Hesse, Germany), transferred to 200 mesh grids, stained with Reynolds’ lead citrate solution, then analyzed and photographed using a transmission electron microscope (JEOL, JEM-1400, Japan) at a current HT 80 KV according to [15 (link)].
Araldite resin
Manufactured by Electron Microscopy Sciences
Sourced in United States
Araldite resin is a two-component epoxy resin designed for a variety of laboratory applications. It is a versatile and widely used material in the field of electron microscopy for embedding and embedding biological and industrial samples prior to sectioning and analysis. The resin provides a stable and inert support matrix for the specimen, enabling high-quality sectioning and preservation of ultrastructural details.
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Lab products found in correlation
Ultracut uct, by Leica (2 mentions)
Jem 1400, by JEOL (1 mentions)
Tecnai t12, by Thermo Fisher Scientific (1 mentions)
Uc6 ultramicrotome, by Leica (1 mentions)
Leo 906 electron microscope, by Zeiss (1 mentions)
Tecnai g2 spirit, by Thermo Fisher Scientific (1 mentions)
Osmium tetroxide, by Merck Group (1 mentions)
Leo 906 e electron, by Zeiss (1 mentions)
6 protocols using araldite resin
1
Ultrastructural Changes in Botrytis cinerea
2
Ultrastructural Imaging of Mouse Cardiac Tissue
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Mice were euthanized and perfusion-fixed with 4% PFA in PBS under anesthesia. Left ventricles were collected and stored in 4% PFA overnight at 4°C. Tissue was trimmed and further fixed in 2% PFA, 2.5% glutaraldehyde, and 2 mM CaCl2 in 0.1 M cacodylate buffer (pH 7.2) at room temperature for 60 min; quenched in 50 mM glycine in 0.1 M cacodylate buffer; and washed and postfixed in 1% (w/v) osmium tetroxide, 0.1 M cacodylate buffer for 30 min, and 0.5% (w/v) osmium tetroxide and 1.5% (w/v) potassium ferrocyanide for 30 min. Specimens were then treated with 1% (w/v) tannic acid for 60 min, washed, en bloc stained with 1% (w/v) uranyl acetate, and dehydrated using 30, 50, 70, 90, and 100% ethanol and 100% acetone in series. After dehydration, specimens were infiltrated and embedded in Araldite resin (Electron Microscopy Sciences) following the manufacturer’s recommendations. Ultrathin sections were cut on a Leica UC6 ultramicrotome (Leica Microsystems Inc.), poststained with uranyl acetate and lead citrate, and examined in an FEI Tecnai T12 transmission electron microscope operated at 80 kV. Digital images were acquired by an AMT bottom-mount charge-coupled device camera and AMT600 software. Tissue samples were prepared and examined in the Electron Microscopy Core Imaging Facility of the University of Maryland School of Dentistry. Two animals were analyzed per group.
3
Viral Particle Identification in Piglet Tissue
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Tissue fragments of the CP and small intestine of one piglet with nonsuppurative meningoencephalitis and vacuolization of apical enterocytes that were collected during autopsy for routine histopathological analysis and maintained in 70% alcohol were selected for TEM processing. This was done primarily to identify viral particles in these tissues, based on our previous findings in newborn piglets with ETNL3 (link). These sections (measuring 1 mm3) were rehydrated in ethanol gradient (50–30° GL) and washed three times in 0,1 M PBS. The samples were fixed in 3% glutaraldehyde in 0.1% cacodylate buffer, post-fixed in 1% osmium tetroxide in 0,1 M cacodylate buffer, dehydrated in ethanol gradient and, after two baths in propylene oxide, embedded in Araldite resin (Electron Microscopy Sciences, Hatfield, PA, USA). Ultrathin sections (60 nm) were obtained by using a ultramicrotome (Leica Ultracut UCT, Solms, Hesse, Germany), transferred to 200 mesh grids, stained with 2% uranyl acetate and lead citrate (Reynold’s solution) or 3% phosphotungstic acid, then analyzed and photographed using a transmission electron microscope (FEI Tecnai G2, FEI Company, Hillsboro, OR, USA).
4
Ultrastructural Analysis of SK-MEL-37 Cells
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SK-MEL-37 cells were starved with EBSS and later fixed in a 2% paraformaldehyde, 2% glutaraldehyde solution (1:1) in sodium cacodylate buffer 0.1 M (pH 7.4) for 24 h at 4°C. The cells were subsequently post-fixed in 1% osmium tetroxide, dehydrated in a graded ethanol series and embedded in Araldite resin (Electron Microscopy Science). Ultrathin sections (~90 nm) were stained with uranyl acetate and lead citrate and examined using a ZEISS Leo 906 electron microscope (Carl Zeiss).
5
Ultrastructural Analysis of Listeria monocytogenes Infection in Macrophages
6
Electron Microscopy of SCC-13 Cells
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After 24 h of the treatments, SCC-13 cells were detached with TrypLE™ Express, washed, and centrifugated at 1000 rpm for 3 min. Then, the pellets were fixed by immersion in Karnovsky mixture containing 2% (v/v) glutaraldehyde and 4% (v/v) formaldehyde in 0.1 M cacodylate buffer plus 7% sucrose. After fixation, cells were washed with 0.1 M cacodylate buffer and post-fixed with 1% osmium tetroxide (Sigma-Aldrich, St. Louis, USA) in the same buffer for 1 h. Subsequently, the pellets were dehydrated in a graded acetone series and embedded in Araldite resin (Electron Microscopy Sciences, Hatfield, PA). Thin sections were cut with a diamond knife on a JEOL JUM-7 ultramicrotome (Nikon, Tokyo, Japan), stained with uranyl acetate and lead citrate, and examined using a Zeiss LEO 906-E electron (Oberkochen, Germany).
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