A Special Word on
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 waranted.
The factors that affect penetration and antigen accessibility
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
With the acchieved 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.
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.
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