Protein Chemistry. Structure, Properties, Research Methods - Shendryk A.N. 2022

Methods for Experimental Investigation of Protein Structure
Electron Microscopy
Preparation and "Staining" of Samples

Sample preparation for Electron Cell/15.html">Microscopy analysis is a highly critical part of the experiment and involves several stages. Biological specimens are prepared in the following sequence.

> The First stage involves fixation: soaking the specimen in a solution such as glutaraldehyde.

> Dehydration.

> Embedding in a plastic block.

> Sectioning the embedded sample into thin slices using a Glass or diamond knife, or an ultramicrotome.

“Staining”. In Electron Microscopy of biological specimens, two MAIN TYPES OF staining are generally used: positive and negative. In positive staining, dye molecules bind to various sites on the macromolecule. In negative staining (negative contrast), macromolecules are embedded in a thin film of stain. Heavy metal salts are typically used as stains.

Staining creates regions of varying electron density, which makes it possible to reveal the surface topography and Structural Features of the sample (see figure below).

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In positive contrasting, the sample is stained in solution and placed onto a grid. In negative contrasting, a drop of the solution containing the sample is applied to a carbon-coated copper grid. The sample molecules are rapidly adsorbed by the film (1–2 min), after which the liquid is carefully removed, and a drop of the “stain” solution is applied. After 15–20 s, the contrasting solution is removed, and the sample is air-dried.



Last update: 06/08/2026

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