Dissected ST and IN samples were immediately fixed in a mixture of 2.5% (w/v) paraformaldehyde (Fluka, Buchs, Switzerland) and 2.5% (v/v) glutaraldehyde (Sigma, Aldrich, St. Louis, MO, USA) [4 (link)] and then post-fixed in 2% (w/v) osmium tetroxide dissolved in 0.05 M sodium cacodylate buffer (Fluka) for 6 h at 4 °C. The samples were progressively dehydrated through a series of ethanol solutions, followed by propylene oxide substitution. They were infiltrated with a graded series of Epon epoxy resin (Sigma-Aldrich, St. Louis, MO, USA) mixtures (for 48 h in total) and the resin was polymerized at 65 °C for 16 h. Semi-thin sections (2.5 µm thick) were taken on an Ultracut E ultramicrotome (Leica, Wetzlar, Germany) and stained for 10 min with 0.1% (w/v) toluidine blue in 1% (w/v) borax. After washing with distilled water, they were examined under a Vanox light microscope (Olympus, Tokyo, Japan) equipped with a Sony ILCE-7 digital camera (Sony, Tokyo, Japan) and an Olympus cellSens Standard ver. 1.7 image analysis system (Olympus).
Epon epoxy resin
Manufactured by Merck Group
Sourced in United States
Epon epoxy resin is a versatile laboratory material used for embedding and sectioning samples in preparation for microscopic analysis. It is a two-part epoxy system that cures to form a hard, durable plastic-like material. The resin's core function is to provide a stable, high-quality support matrix for delicate specimens, enabling thin sectioning and detailed observation under a microscope.
Automatically generated - may contain errors
Lab products found in correlation
Glutaraldehyde, by Merck Group (3 mentions)
Ultracut e ultramicrotome, by Leica (2 mentions)
Vanox light microscope, by Olympus (1 mentions)
Polysine slide, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Lr white acrylic resin, by Merck Group (1 mentions)
3 protocols using epon epoxy resin
1
Ultrastructural Analysis of Plant Tissues
2
Cell Suspension Fixation and Embedding
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Two-week-old cell suspensions were sampled and fixed in 2% (w/v) paraformaldehyde (Fluka, Buchs, Swiss) and 3% (v/v) glutaraldehyde (Sigma, St. Louis, MO, USA) in 0.05 M sodium cacodylate buffer (Fluka) at pH 7.2 at room temperature for 4 h. The samples were rinsed three times in the same buffer, and post-fixed in 2% (w/v) OsO4 (Carl Roth, Karlsruhe, Germany) in 0.05 M cacodylate buffer at 4 °C for 6 h. After rinsing in 0.05 M cacodylate buffer, explants were dehydrated in a graded ethanol series (in 10% increments), followed by absolute ethanol and propylene oxide. The samples were infiltrated in graded EPON epoxy resin (Sigma) mixtures for 48 h in total. The samples were transferred to flat embedding molds, and the resin was polymerized at 65 °C for 16 h. Semi-thin (2 µm) sections were cut using a Leica Ultracut E ultramicrotome (Leica, Wetzlar, Germany). Semi-thin sections were stained with aqueous 0.1% (w/v) toluidine blue in 1% (w/v) sodium tetraborate solution and were analyzed by means of a light microscope (see above).
3
Ultrastructural Analysis of Root Syncytia
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Root segments with syncytia were fixed in 4% (w/v) paraformaldehyde (Sigma-Aldrich) and 0.25% (v/v) glutaraldehyde (Fluka) in 50 mM phosphate buffered saline (PBS, pH=7.0) for 2 h at room temperature. Afterwards, they were washed three times in 50 mM PBS for 15 min and embedded in Lowicryl K4M acrylic resin (Sigma-Aldrich) using a modified PLT technique (Table 1, Edelmann, 2001, modified) . The procedure was based on a stepwise lowering of temperature throughout the
dehydration process, infiltration, embedding and polymerization of acrylic resin. For comparison a batch of samples was fixed in 2% (v/v) glutaraldehyde and 2% (w/v) paraformaldehyde in 50 mM PBS for 2 h at room temperature, rinsed three times in 50 mM PBS, dehydrated, infiltrated and embedded in LR-White acrylic resin (Sigma-Aldrich). Resin blocks were polymerized at 48 °C for 48 h and serially sectioned on semithin (3 μm thick) cross sections that were collected on slides coated with poly-Llysine (Polysine® slides, Thermo Scientific, Walthman, MA, USA). Ultrathin (~80 nm thick) cross sections were collected on formvar-coated nickel grids. For comparative anatomical and ultrastructural studies few samples were also embedded in Epon epoxy resin (Sigma-Aldrich) according to procedure described by Grundler et al. (1998) .
dehydration process, infiltration, embedding and polymerization of acrylic resin. For comparison a batch of samples was fixed in 2% (v/v) glutaraldehyde and 2% (w/v) paraformaldehyde in 50 mM PBS for 2 h at room temperature, rinsed three times in 50 mM PBS, dehydrated, infiltrated and embedded in LR-White acrylic resin (Sigma-Aldrich). Resin blocks were polymerized at 48 °C for 48 h and serially sectioned on semithin (3 μm thick) cross sections that were collected on slides coated with poly-Llysine (Polysine® slides, Thermo Scientific, Walthman, MA, USA). Ultrathin (~80 nm thick) cross sections were collected on formvar-coated nickel grids. For comparative anatomical and ultrastructural studies few samples were also embedded in Epon epoxy resin (Sigma-Aldrich) according to procedure described by Grundler et al. (1998) .
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