9.3 Protein Localization in Conventional TEM

Figure 9.11: Pros and Cons in Protein Labelling Methods

The above figure shows some pros and cons of some common protein localization methods in a conventional TEM.

9.3.1 Immuno-gold EM

This is the labeling of proteins with specific antibodies (much like in fluorescence light microscopy):

Figure 9.12: Gold-Labeling Proteins

Here, gold is attached to an a(n) (in)direct antibody. Colocalization analysis is also possible with two different antibodies and different gold particle sizes.

9.3.1.1 Labeling Golgi Proteins

Figure 9.13: Immuno-Labeling of a Post-Embedded Protein

Labelling in post-embedded proteins is generally carried out on sections (i.e., Tokuyasu sections) - many different combinations of antibodies can be carried out on one sample. No detergents are also needed, and antibodies can only detect antigens that are exposed to the surface of the section.

Figure 9.14: Immuno-Labeling of a Pre-Embedded Protein

Pre-embedded proteins are labelled inside a cell. Because of this, permeabilization with proteins is needed; silver enhancements are also required to detect small gold particles.

9.3.2 APEX2

Figure 9.15: The APEX2 Protein

APEX2 is a peroxidase that is active in the cytosol. Itcan also be fused to any proteins of interests - this fusion can also be expressed in cells.

Cells are chemically stained and processed with TEM imaging (either 2D or 3D).

9.3.3 Some Comparisons Between Immuno-Gold Labeling and Other Staining Techniques

Figure 9.16: Comparisons Between Immuno-Gold Labeling and Other Staining Techniques

The above table lists some differences between immuno-gold labeling and other staining techniques.