For most applications the detection of ultra small gold particles in electron microscopy requires a particle enhancement procedure. Danscher’s method has always been the standard for this purpose and for a long time attempts to improve the features of Danscher’s system met with only limited success.
The goal in the development of AURION R-Gent SE-EM was to create a new system with enhancement efficiency and homogeneity at least comparable to Danscher’s method, but with reduced acidity and light sensitivity. In addition, the reagents should have low viscosity for the suitability in pre-embedding immunogold labeling.
The silver enhancement reaction is a gold particle-catalyzed reduction in which silver ions are reduced to metallic silver with a photographic developing compound as the electron source. In addition in many applications a “protective colloid” is added to the enhancement solution to limit diffusion of reagents to the gold particle surface, thus providing a means for controlling particle growth.
AURION R-Gent SE-EM is a high efficiency silver enhancement reagent for electron microscopy. It intensifies the gold particles by homogeneous deposition of metallic silver on the particle surface. The resulting electron dense signal is easily detected and is compatible with heavy metal counterstaining. The reagent has extremely delayed auto-nucleation and can be used under standard laboratory light conditions. It also has low viscosity, which is especially advantageous for pre-embedding immunogold applications. The enhancement mixture has a pH of 8.1-8.2.
AURION R-Gent SE-EM has been tested intensively for the enhancement of AURION Ultra Small Immunogold reagents.
AURION R-Gent SE-EM is available in a kit containing the following:
- 30 ml or 90 ml of ready-to-use ENHANCER,
- 3 ml of concentrated INITIATOR and
- 30 ml of ACTIVATOR.
- a 3 ml empty dropping bottle, labeled “DEVELOPER”.
The INITIATOR is a concentrated solution which must be diluted and activated before use, using the ACTIVATOR. This resulting mixture is the DEVELOPER.
The now ready-to-use DEVELOPER has a shelf life of one month. It is suggested to prepare fresh DEVELOPER at least every month.
The undiluted solutions have a shelf life of 10 months when stored at 4°C. INITIATOR can be stored at -20°C for prolonged shelf life.
For enhancement 1-2 drops of DEVELOPER are mixed with 20 drops of ENHANCER. The typical enhancement time is between 20 and 40 minutes. Specimens may be contrasted according to standard procedures.
The kit with 30 ml of ENHANCER accomodates up to 1000 EM grid specimens. The kit with 90 ml of ENHANCER is suitable for pre-embedding immunogold labeling; the number of specimens it accomodates depends on the volume of reagent used per specimen.
For each lot, the specific enhancement activity and level of autonucleation are monitored by spectrophotometric techniques.
"...silver enhancement reaction is a gold particle-catalyzed reduction in which silver ions are reduced to metallic silver with a photographic developing compound as the electron source..”
Detailed information is provided on the package inserts.
Post-embedding enhancement (on-grid)
Place grids on drops of the enhancement mixture arrayed on a sheet of Parafilm™.
Incubate specimens (e.g. vibratome sections) in Petri dishes or Eppendorf tubes containing the enhancement mixture, with agitation. When enhancement is completed,
the specimens are washed with distilled water. A fixation step with photographic fixer is not required.
The AURION R-Gent SE-EM components are stored at 4°C and allowed to reach room temperature before use. SE-EM INITIATOR may be stored at -20°C for prolonged shelf life.
Desmin labeling in heart muscle with Ultra Small Gold conjugate and AURION R-Gent SE-EM. Courtesy Prof. Dr. Müller Höcker, University Munich, FRG.