AURION IMMUNOGOLD REAGENTS EDITION VI PRODUCT INFORMATION


zzz ECS – Enhancement Conditioning Solution


Aurion’s Enhancement Conditioning Solution (ECS) is a concentrate that requires a 10x dilution with distilled grade water before use. It helps optimise enhancement by conditioning specimens labelled with (ultra small) gold conjugates before the silver enhancement procedure using Aurion R-Gent SE- EM. Aurion ECS is a prerequisite to condition enhanced specimens for a second incubation series in double labelling experiments (Yi et al, 2001, Journal of Histochemistry and Cytochemistry 49(3): 279-283).


Introduction


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 enhance- ment solution to limit diffusion of reagents to the gold particle surface, thus providing a means for controlling particle growth.


Product Description


Incubation buffers suited for immuno gold labelling commonly contain ions such as phosphate and chloride which are not compatible with silver ions causing precipitation of insoluble silver salts. These ions therefore need to be thoroughly removed before enhancement. Whereas pure water is suited to fulfil this requirement in the purely chemical sense, it is rarely the best way when working with delicate specimens potentially affecting ultrastructural details vulnerable to drastic changes in ionic concentration. Using water it is also necessary to carefully blot the specimens and remove the water without drying before applying Aurion R-Gent SE-EM. Failing to do so will result in dilution and diminished activity of the silver enhancement reagents, especially when working with small volumes.


Aurion developed Aurion ECS to negotiate optimum conditions for enhancement while at the same time avoiding the risks posed by water. Its ionic strength is similar to immuno incubation buffers and its make up is compatible both with these buffers as well as with Aurion R-Gent SE-EM.


Aurion ECS is therefore highly recommended for single labelling experiments. It is an absolute requirement for double labelling where ECS not only reconditions specimens for the next immuno steps but also avoids the use of aldehyde fixation after the first immuno gold silver labelling sequence.


TECHNICAL TIPS... Application Instructions


Detailed information is provided in the package insert.


Used as a 10x dilution Aurion ECS serves as a washing medium after immuno incubation and before silver enhancement (immuno procedure – wash – silver enhancement).


In addition to this application in singe labelling it is required in double labelling experiments as a washing medium between the first silver enhancement step and the second immuno incubation sequence. (immuno incubation1 – wash – silver enhancement1 – wash – immuno incubation 2 – wash – silver enhancement 2).


Storage


Aurion ECS has a guaranteed shelf life of 18 months from the date of quality control analysis. Store at 4-8°C. Do not freeze.


Additional Information


For additional information, please see the Aurion Immunogold Newsletters and Technical Tips on our website.


AURION R-Gent SE-EM Application Example


ER exit site in 60 nm-thin cryosection of Hepg2 cells, labeled for COPII (primary antibody against sec23 was obtained by ABR) and detected with Fab-goat-anti-rabbit, conjugated to ultra-small gold, silver enhanced for 30 minutes (from Aurion).


The arrows point to labeled COPII-coats on vesicular and tubular membranes, which are located close to the ER.


The information of a thin section is not sufficient to conclude how the membranes are related to each other- if they are still connected to the ER, or if they are free.


Therefore we performed 3D electron tomography on 400nm thick cryosections, which were labeled similar for COPII. (see next picture).


ER = endoplasmic reticulum, PM = plasma membrane, MVB = multi-vesicular body, Bar = 100 micrometer


2 views of a model of a COPII-labeled ER-exit site, resolved from 400nm thick cryo-sections of Hepg2 cells, labeled like described for the ultrathin section before.


Note that the labeling for COPII is assessable throughout the section.


ER = light blue, Free membrane carriers of vesicular and tubular shape, partially labeled for COPII = yellow, COPII = silver enhanced-red


Courtesy of: Dagmar Zeuschner, Judith Klumperman (Department of Cell Biology, UMC Utrecht, The Netherlands) and Willie Geerts, Abraham Koster (Molecular Cell Biology, Utrecht University, The Netherlands)


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