Double labeling with two Ultra Small Reagents

Aurion has once more confirmed its leading role in immunogold technology through a publication in the Journal of Histochemistry and Cytochemistry 49(3): 279-283 (2001).

In this publication it was demonstrated for the first time in history that it is possible to perform a double labeling experiment using two ultra small gold conjugates and differential silver enhancement using Aurion R-Gent SE-EM, the enhancement reagent of choice for electron microscopy applications.

This approach was successful thanks to the excellent and tuned qualities of the reagents involved:

• the high penetration characteristics of Aurion's Ultra Small Immunogold Reagents,
• the low viscosity and high efficiency of the silver enhancement reagents and
• the excellent homogeneity of the resulting silver particles, providing the basis to distinguish particle sizes as dependent on silver enhancement times.

One more item was required: the Enhancement Conditioning Solution (ECS)

What is ECS intended for?

ECS is used as a bridge solution between the gold incubation and the silver enhancement.

Before the silver enhancement step in a single labeling the specimen is fixed with glutaraldehyde and washed with distilled water. The fixation prevents ultrastructural defects that would result from the water wash of a mildly fixed vibratome section. The water wash step is necessary for compatibility of the silver enhancement step.

Obstacles in Double Labeling

When performing a double labeling with ultrasmall gold conjugates a few obstacles have to be overcome:

• a fixation step after the first labeling before silver enhancing can not be performed as this would make it impossible to do a second labeling,
• Washing the specimen in distilled water without a fixation step affects the morphology of the specimen in a negative way
• Washing in incubation buffer is not compatible with silver enhancement.

Aurion developed, in close co-operation with Hong Yi from Emory University, Aurion ECS (Enhancement Conditioning Solution) which replaces the fixation and water wash step. The product is compatible with buffer and silver enhancer and does not affect morphology.

Aurion ECS in Double Labeling

Schematically this is the sequence of steps for double labeling using ultrasmall gold conjugates:

Labeling 1:

• gold incubation
• washing in incubation buffer
• ECS
• silver enhancement
• ECS + thiosulphate
• ECS

Labeling 2:

• gold incubation
• washing in incubation buffer
• ECS
• silver enhancement

Please note that Aurion ECS can only be used with Aurion R-Gent SE-EM, because the components of ECS are tuned to the components of R-Gent SE-EM.

Aurion as well as leading researchers in the field are convinced that these new possibilities will facilitate and simplify immuno electron microscopic research in the bio-medical field.