A Special Word On Pre-Embedding

Pre-embedding labeling of dopamine transporter in rat brain with single Fab Ultra Small Immunogold and R-Gent SE-EM. Penetration at 8 µm from the section surface. Courtesy Mrs. Hong Yi, Emory University Atlanta

The introduction of Ultra Small gold conjugates and AURION R-Gent SE-EM silver enhancement for electron microscopy has lead to a breakthrough in the feasibility of pre-embedding procedures. Due to their reduced size, the gold conjugates have a high detection sensitivity and are less prone to steric hindrance. They penetrate more readily without pre-treatment with detergents. From the specimen side the accessibility and preservation of antigens should be warranted.

The factors that affect penetration and antigen accessibility are:

the specimen properties (fixation, degree of cross-linking, dimensions, chemical and physical composition, degree of permeabilization),
the characteristics of the reagents (Single Fab, F(ab’)2 vs. intact immunoglobulin, gold particle size),
the incubation conditions (ionic strength, pH, di-electric constant, temperature, time).

To come to a successful result these different parameters should be in balance (e.g. limited cross-linking may tolerate short incubation times, extensive cross-linking will require longer incubation times). In addition these parameters have to be balanced against several other demands such as ultrastructural integrity. Therefore the use of detergents is not always acceptable.

With the achieved subnanometer particle size, Aurion has focused its attention for further improvements on the size of the protein part in the secondary reagents. The size of the immunogold conjugate naturally also depends on the size of the coupled binding agent (i.e. the secondary antibodies). To this end both F(ab’)2 fragments and single Fab fragments were prepared and coupled to ultra small gold particles. In comparison with the intact immunoglobulin conjugates the F(ab’)2 conjugates show a significantly higher label density and label efficiency, whereas a further improvement is found with the Fab conjugates. Although single Fab conjugates at least theoretically will diffuse more readily, this benefit is counteracted by the fact that only one binding site is left per conjugate: their binding is dependent on the affinity of the single binding site.

*Pre-embedding labeling of parvalbumin in rat brain with Single Fab Ultra Small Immunogold and R-Gent SE-EM. Courtesy Mrs. Hong Yi, Emory University, Atlanta*

The incubation conditions are the final factor of importance in pre-embedding labelling. In general ultra small gold particle labeled antibodies diffuse at lower rates than those based on other markers like fluorescent labels; the main reason may be that the negative charges on the gold particle surface are repulsed by negative charges in the tissue. Therefore incubation times generally should be longer. This repulsion is even more prominent in aldehyde fixed specimens where many of the amino groups will have been modified. In AURION Newsletter nr. 1 these charge-based phenomena are more extensively discussed and related to background phenomena.
Prolonged incubation with immunoreagents also implies that the time of washing will have to be prolonged for efficient removal of unbound reagents.