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The Immunogold Silver Staining technique (IGSS) finds application both at the electron microscope and the light microscope level. In addition the technique can be equally 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.
Product Description 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 30 minutes. Auto-nucleation becomes visible only after 40-60 minutes. Light microscopical specimens may be counterstained according to standard procedures. The enhancement mixture has a pH value of 8.3-8.5.
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.
The AURION R-GENT SE-LM components are stored at 4°C and allowed to reach room temperature before use.
R-Gent SE-LM for Light Microscopy 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. Bio Assays
Light microscopy evaluation of tubulin labeling with Ultra Small Immunogold Reagents and silver enhancement.
Upper panel: bright field mode Lower panel: epi-polarization mode
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.
For additional information please see the Aurion Immunogold Newsletter and Protocol sections.
COL-AURION: Colloidal Gold Based Protein Stain
Col-Aurion is a colloidal gold particle based total protein stain, developed for the sensitive staining of electrophoretically separated protein bands on nitrocellulose or PVDF™ blotting membranes. Colloidal gold particles accumulate at the site of the protein bands on the membrane, generating an intense dark red staining pattern. The total protein stain assists in assessing immunoblotting results and to evaluate the effectivity of the blotting procedure.
The negative surface charge of colloidal gold particles is responsible for their high affinity for positively charged macromolecules. This characteristic was the basis for the development of a total protein stain based on colloidal gold.
Product Description Col-Aurion is a total protein stain consisting of a solution of coated gold 80
particles with an average particle diameter of 15 nm. The staining principle is based on the electrostatic binding of negatively charged gold particles to proteins with a positive charge present at low pH.
A unique feature of Col-Aurion is the use of BSA-c™ to shield off the surface of the gold particles. Destabilization of gold particles that might occur as a result of interaction with detached protein is thus prevented. The strong negative charge of BSA-c™ gives an additional increase in sensitivity of the stain.
Col-Aurion total protein stain has a pH of 3.2 and is ready to use. For removal of surplus of weakly bound protein from the membrane, a 10 ml vial of Tween-20™ is included.
Col-Aurion has a guaranteed shelf life of 18 months from the date of quality control analysis. Store at 4-8°C. Do not freeze.