Spheroids were fixed and processed as previously described52 (link). Samples were fixed for 1 hour at room temperature in a fixative solution containing 4% Paraformaldehyde (EMS), 2% Glutaraldehyde (EMS), and 0.1M Na Cacodylate (EMS). Samples were then osmicated, stained with uranyl acetate and embedded in EMbed 812 (EMS). Ultrathin sections (120 nm) from each spheroid sample were observed with a FEI Helios NanoLab™ 660 FIBSEM using extreme high resolution (XHR) field emission scanning electron microscope equipped with a Concentric (insertable) higher energy electron detector, all images were taken using 4 Kv and 0.2 current landing voltage at high magnifications (15000–35000x).
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Ultrastructural Analysis of 3D Spheroids
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Cacodylate
Electron
Fixative
Glutaraldehyde
Paraformaldehyde
Scanning electron microscope
Uranyl acetate
Corresponding Organization :
Other organizations : Case Western Reserve University, University School, George Washington University, New York Stem Cell Foundation
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Variable analysis
independent variables
- None explicitly mentioned
- Ultrastructural morphology of spheroid samples observed using FEI Helios NanoLab™ 660 FIBSEM at high magnifications (15000–35000x)
- Fixation time (1 hour)
- Fixative solution composition (4% Paraformaldehyde, 2% Glutaraldehyde, 0.1M Na Cacodylate)
- Osmication, staining with uranyl acetate, and embedding in EMbed 812
- Ultrathin sections (120 nm) from each spheroid sample
- Microscope settings (4 Kv and 0.2 current landing voltage)
- No positive or negative controls were explicitly mentioned in the provided information.
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