The Immunogold Silver Staining technique (IGSS) finds application both at the electron microscope and the light microscope level. In addition the technique is used to identify proteins or nucleic acids after blotting.
Light microscopical and macroscopical visualization of the latent gold signal requires an enhancement system that renders a high contrast signal. Light insensitivity and negligible autonucleation are required for ease of handling and low background. AURION R-Gent SE-LM is a two-component reagent that meets these requirements.
The silver enhancement reaction is based on the gold particle catalyzed reduction of Ag+ to metallic silver using photographic developing compounds as the electron source. For light microscopy and immunoblotting applications the generated silver signal should be of high contrast. Furthermore the signal should be permanent and compatible with counterstaining.
The AURION R-GENT SE-LM components constitute a Silver Enhancement Reagent which increases the average gold cluster or particle size by deposition of metallic silver facilitating detection at the light microscopical level. The generated brown-black signal is also easily detected in bio assays and is compatible with counterstaining in light microscopy. AURION R-GENT SE-LM has been tailored for the enhancement of AURION Ultra Small Immunogold reagents and is equally suited for the larger sized particles in the AURION Conventional Immunogold reagents.
For enhancement, equal amounts of the DEVELOPER and ENHANCER are mixed well and applied to the specimen. The enhancement mixture is easy-to-use, exhibits extremely delayed auto-nucleation and can be used under standard laboratory light conditions. Typical enhancement times are between 15 and 25 minutes. Auto-nucleation becomes visible only after 40 minutes. Light microscopical specimens may be counterstained according to standard procedures. The enhancement mixture has a pH value of 8.2-8.6.
AURION R-GENT SE-LM is available as a kit in two unit sizes (2 x 30 ml or 2 x 250 ml) and consists of a separate DEVELOPER and ENHANCER. The supplied amounts accomodate 600 and 5000 LM specimens respectively at 100 µl/specimen, or 60 and 500 bio assay specimens at 1 ml/specimen. The reactivity is tested on dot-spots and the absence of autonucleation is monitored by spectrophotometric techniques.
Light microscopy evaluation of tubulin labeling with Ultra Small Immunogold Reagents and silver enhancement. Upper panel: bright field mode Lower panel: epi-polarization mode
Detailed information is provided on the package inserts.
Slide or Coverslip labeling
A few drops of the freshly prepared enhancement mixture are applied to cover the specimen. During enhancement the specimens are kept in a moist chamber. The ongoing enhancement may at intervals be monitored with an inverted light microscope. When enhancement is judged to be complete, the specimens are washed with distilled water. A fixation step with photographic fixer is not required.
Whole mount labeling
Specimens like floating (vibratome) sections can be enhanced in Petri dishes or 6-24 well plates.
Depending on the type of assay the enhancement for bio assays can be performed in sealed plastic bags, Petri dishes or in disposable screw cap sealed tubes.
The AURION R-GENT SE-LM components are stored at 4°C and allowed to reach room temperature before