التفاصيل البيبلوغرافية
| العنوان: |
Sugar and ice: Immunoelectron microscopy using cryosections according to the Tokuyasu method. |
| المؤلفون: |
Möbius, Wiebke, Posthuma, George |
| المصدر: |
Tissue & Cell; Apr2019, Vol. 57, p90-102, 13p |
| مصطلحات موضوعية: |
IMMUNOELECTRON microscopy, MICROSCOPY, ELECTRONIC probes, ORGANELLES, ORGANIC solvents, ELECTRON microscopy |
| مستخلص: |
Highlights • Versatile method for preparation of cryosections for immunolabeling of fixed samples. • Target molecules are accessible to antibodies from the section surface. • Antigenicity is preserved by mild fixation and by keeping an aqueous environment. • Correlative light and electron microscopy workflows are established. • The use of particulate markers allows quantification and statistical evaluation. Abstract Since the pioneering work of Kiyoteru Tokuyasu in the 70ths the use of thawed cryosections prepared according to the "Tokuyasu-method" for immunoelectron microscopy did not lose popularity. We owe this method a whole subcellular world described by discrete gold particles pointing at cargo, receptors and organelle markers on delicate images of the inner life of a cell. Here we explain the procedure of sample preparation, sectioning and immunolabeling in view of recent developments and the reasoning behind protocols including some historical perspective. Cryosections are prepared from chemically fixed and sucrose infiltrated samples and labeled with affinity probes and electron dense markers. These sections are ideal substrates for immunolabeling, since antigens are not exposed to organic solvent dehydration or masked by resin. Instead, the structures remain fully hydrated throughout the labeling procedure. Furthermore, target molecules inside dense intercellular structural elements, cells and organelles are accessible to antibodies from the section surface. For the validation of antibody specificity several approaches are recommended including knock-out tissue and reagent controls. Correlative light and electron microscopy strategies involving correlative probes are possible as well as correlation of live imaging with the underlying ultrastructure. By applying stereology, gold labeling can be quantified and evaluated for specificity. [ABSTRACT FROM AUTHOR] |
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| قاعدة البيانات: |
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