The Aurion reagents may find application both in immunohisto- and cytochemistry and in in situ hybridisation.
Blocking Reagents and Additives
In a labeling experiment, background staining can be reduced by blocking steps before the introduction of immuno-reagents and by compounds added to the immuno-incubation media and washing solutions. Aurion’s blocking reagents and additives include ready-to-use Blocking Solutions, individual components for commonly used blocking solutions, and a proprietary incubation media additive, BSA-c™.
AURION Blocking Solutions are available in different specificities corresponding to the gold conjugate selected. AURION BSA-c™ is strongly recommended for preventing charge based background.
Conjugates and their Gold Particle Sizes
Ideally, markers for highly specific detection compounds should have no influence on their performance. Although it was initially believed that the adsorption of antibodies and other detection proteins onto the gold particles had little or no bearing on their functionality, it was occasionally found that the activity of some reagents was severely impaired as a result of molecular deformation and steric phenomena on the gold particle surface. In addition it has become obvious that the gold particle size influences the efficiency of the labeling: the larger the particle the lower the number of particles found per unit area of specimen. This illustrates the importance of small particles, which have a far smaller influence on the molecule they are coupled to and which take up less space on the sample surface. Aurion offers both Conventional Immunogold Reagents and Ultra Small Immunogold Reagents.
Conventional Immunogold Reagents
Conventional Immunogold Reagents do not require any enhancement procedure and the particles are readily detectable by Transmission and Scanning Electron Microscopy. They are a good choice when the antigen is abundant and the accessibilty of the antigen is relatively good. AURION Conventional Immunogold Reagents are prepared so that overlap does not exist between consecutive sizes (e.g. 6 and 10 nm). However, to facilitate visual discrimination, the combination of two non-consecutive sizes (e.g. 6 and 15 nm or 10 and 25 nm) is recommended for double labeling.
Ultra Small Immunogold Reagents
Significantly higher sensitivity and penetration can be obtained with Ultra Small Immunogold Reagents, which are prepared with subnanometer gold particles. Such particles have far less influence on the adsorbed antibodies or detecting compounds and consequently the reagents behave more as if uncoupled. The smallest possible reagents should always be chosen for those applications where penetration can or should be fully exploited (in pre-embedding labeling, in hydrated specimens like cryosections and in labeling for light microscopy), or where maximum detection levels need to be achieved (low antigen levels). In conjunction with the highly efficient and easy-to-use R-Gent SE-LM and SE-EM silver enhancement reagents, the Ultra Small Immunogold conjugates are the best choice for any application.
Conjugates and their Proteins
One of the most frequently asked questions is ‘which conjugate to choose?’ This choice should be determined by the required resolution and penetration (pre- vs. post-embedding labeling) and how the final signal is evaluated. For instance, the highest resolution is obtained with the smallest possible conjugate and the fewest possible labeling steps. This is an adequate approach when the epitope needs to be localized at the molecular level, for which a one-step labeling with single Fab fragments or ligands coupled to Ultra Small or 6 nm gold particles may provide the best result. (For more detail, refer to information on the Custom Labeling page)
Most post-embedding immunolabeling experiments are performed on sections or replicas, where a two-step labeling is generally the method of choice. Protein A and protein G conjugates bind to limited areas of primary antibodies of selected species with a maximum of one gold particle per primary antibody, thus providing maximum resolution in two-step labeling. Secondary antibody conjugates recognize a larger number of sites on species specific primary antibodies, in general leading to one to four gold particles per primary antibody.
Three-step labeling methods usually are only used in light microscopy detection, where resolution in bright field mode is of less importance than signal intensity.
Silver Enhancement Reagents
The size of Ultra Small gold particles can be increased by silver enhancement allowing their visualization at electron and light microscopy levels. This process involves the gold particle mediated reduction of silver ions and deposition of metallic silver on the particle surface.
Aurion provides two types of silver enhancement reagents, AURION R-Gent SE-EM and AURION R-Gent SE-LM.
AURION R-Gent SE-EM is a high efficiency silver enhancement reagent for electron microscopy applications. It is characterized by its homogeneous enhancement, light insensitivity, low viscosity, near neutral pH and virtually no autonucleation.
AURION R-Gent SE-LM, can be used for light microscopy and blotting applications. It produces a brown to black enhancement when viewed by bright field light microscopy. Furthermore, it is light insensitive, has near neutral pH and extremely low autonucleation.