HIEs (3D) embedded in Matrigel were fixed in a solution of 2% paraformaldehyde + 2.5% glutaraldehyde + 2 mmol/L calcium chloride in 0.1 mol/L cacodylate buffer (pH = 7.4) for 5–7 days at 4°C. They were postfixed in 1% osmium tetroxide in 0.1 mol/L cacodylate buffer for 1 hour and stained en bloc with saturated aqueous uranyl acetate. After a routine dehydration sequence, tissue pieces gradually were infiltrated in a gradient series of Spurr’s Low Viscosity resin (Sigma-Aldrich) and ethanol, then embedded in fresh Spurr’s resin and polymerized at 60°C for 3 days. Thin sections (55–60 nm) were cut on a Leica (Wezlar, BRD) UC7 ultra-microtome using a Diatome Ultra 45 diamond knife. Sections were viewed on a Hitachi (Tokyo, Japan) H7500 transmission electron microscope set to 80 kV. Images were collected using an AMT XR-16 digital camera and AMT Image Capture (Advanced Microscopy Techniques Corp, Woburn, MA), v602.600.51 software.
Spurr s low viscosity resin
Manufactured by Merck Group
Spurr's Low Viscosity resin is a laboratory material used in the preparation of samples for electron microscopy. It is a low-viscosity, epoxy-based resin designed to provide a suitable medium for embedding and supporting specimens during the sample preparation process.
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2 protocols using spurr s low viscosity resin
1
Ultrastructural Analysis of 3D HIEs
2
Microscopic Examination of Barley Caryopses
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For light microscopy, the cross-sections (2 mm) of barley caryopses were fixed in 2% (v/v) glutaraldehyde and 2% (v/v) formaldehyde in 50 mm cacodylate buffer, pH 7.2, overnight at 8°C. After washing, samples were dehydrated in a graded ethanol series following by embedding in Spurr’s low viscosity resin (Sigma Aldrich). Samples were sectioned (1 μm) on a Reichert-Jung Ultracut S microtome (Leica), stained with 2% crystal violet (w/v), and examined with a Zeiss Axio Imager light microscope (Carl Zeiss).
For electron microscopy, Spurr embedded probes were sectioned at 70 nm, collected on 75 mesh copper grids, and stained with uranylacetate and lead citrate35 (link). Samples were examined using Tecnai20 electron microscope (FEI) at 120 kV. Digital recordings were made with a Megaview III (Soft Imaging Systems).
For electron microscopy, Spurr embedded probes were sectioned at 70 nm, collected on 75 mesh copper grids, and stained with uranylacetate and lead citrate35 (link). Samples were examined using Tecnai20 electron microscope (FEI) at 120 kV. Digital recordings were made with a Megaview III (Soft Imaging Systems).
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