Sugar and ice: Immunoelectron microscopy using cryosections according to the Tokuyasu method

Area of Science:

• Cell Biology
• Microscopy Techniques

Background:

• The Tokuyasu-method, developed in the 1970s, is a widely used technique for immunoelectron microscopy.
• It allows for the visualization of subcellular structures using gold-labeled antibodies.

Purpose of the Study:

• To explain the procedure of sample preparation, sectioning, and immunolabeling using the Tokuyasu-method.
• To discuss recent developments and the rationale behind the protocols, including historical context.

Main Methods:

• Cryosections are prepared from chemically fixed and sucrose-infiltrated samples.
• Immunolabeling is performed using affinity probes and electron-dense markers on hydrated sections.
• Antibody specificity is validated using knock-out tissue and reagent controls.

Main Results:

• The Tokuyasu-method preserves antigenicity by avoiding organic solvents and resin embedding.
• It allows antibodies to access target molecules within dense cellular structures.
• Correlative microscopy and stereology enable quantification and evaluation of labeling specificity.

Conclusions:

• The Tokuyasu-method provides high-resolution ultrastructural details of cellular components.
• It remains a valuable technique for immunoelectron microscopy due to its preservation of antigenicity and accessibility.
• Recent advancements enhance its utility for complex correlative and quantitative studies.