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AURION IMMUNOGOLD REAGENTS EDITION IV PRODUCT INFORMATION


zzz R-Gent SE-EM Silver Enhancement Reagents


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.


Introduction TECHNICAL TIPS...


Application Instructions Detailed information is provided in the package inserts.


Electron Microscopy


Post-embedding enhancement (on-grid) Place grids on drops of the enhancement mixture arrayed on a sheet of Parafilm™.


Pre-embedding enhancement


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.


Light Microscopy On-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


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.


Storage


The AURION R-Gent SE-EM components are stored at 4°C and allowed to reach room tem- perature before use. SE-EM INITIATOR may be stored at -20°C for prolonged shelf life.


Additional Information


For additional information please see the Aurion Immunogold Newsletter and Protocol sections


8


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.


Product Description


AURION R-Gent SE-EM is a high efficiency silver enhance- ment 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 counter- staining. 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 enhance- ment mixture has a pH of 8.1-8.2.


AURION R-Gent SE-EM has been tested ntensively 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.


"...silver enhancement reaction is a gold particle-catalyzed reduction in which silver ions are reduced to metal- lic silver with a photographic developing compound as the electron source..."


Desmin labeling in heart muscle with Ultra Small Gold conjugate and AURION R-Gent SE-EM. Courtesy Prof. Dr. Müller Höcker, University Munich, FRG.


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 one drop of DEVELOPER is mixed with 20 drops of ENHANCER. The typical enhance- ment time is between 20 and 40 minutes. Specimens may be contrasted according to standard proce- dures.


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 accomo- dates depends on the volume of reagent used per specimen.


For each lot, the specific enhancement activity and level of autonucleation are monitored by spec- trophotometric techniques.


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