SPI-Mark™ Silver Enhancement Kit

For light and electron microscopy

LM/EM Silver Enhancement Kit

The SPI-Mark Silver Enhancement kits provide simple to use and sensitive systems for the amplification of immunogold labeled samples in light microscopy, electron microscopy, and blotting applications. Silver enhancement occurs during the reduction of silver from one solution (e.g. the Enhancer) by another (e.g. the Initiator) in the presence of gold particles. The reduction reaction causes silver to build up preferentially on the surface of the gold particles which are conjugated (e.g. attached) to antibodies, proteins or other macromolecules on the target specimen.

The silver enhancement is rapid but easily controllable and within a comfortable time span (e.g. minutes). Amplification of the gold signal by 10-100X is readily (and routinely) achieved. The reaction is insensitive to light, is simply stopped by washing in water, and needs no fixing. The learning curve for doing this kind of work tends to be longer for most researchers than EM techniques generally, and we have tried to put together a few hints to shorten the length of the learning curves.

Light Microscopy

Specimens may be immunogold labeled sections of tissue, whole cells, smears, chromosomes, etc. mounted on glass slides, cell monolayers or tissue slices in culture. Small gold particles are not visible in the LM because of the limit of resolution (> 400nm) of the microscope. After the immunogold labeling the specimen is washed in water and simply enhanced in a single step with the solutions provided. The final result is an intense brown/black stain at the site of the gold label. See SPI-Mark Gold Probes for Light Microscopy.

Both 1nm and 5nm immunogold labels give excellent results with silver enhancement, 1nm gold labels being of particular value when penetration of cells is required. Adequate amplification is usually obtained within a period of 5-10 minutes and may be monitored on the microscope during the reaction. The enhancement is performed by mixing together one drop of each solution and applying to the slide.

The reaction can be performed with a cover slip in place to allow high magnification viewing, even with oil immersion. The reaction is stopped by simply washing in water. It may be continued if necessary with fresh solutions.

The LM/EM Silver Enhancing Kit is sufficient for at least 300 incubations on glass slides. After washing, the slides may be counterstained and mounted as normal. Because of the discrete nature of the growth of silver on the gold particles, no diffusion of the signal occurs. The silver stain produces a permanent, non fading label of sharp resolution and high contrast in bright field viewing.

Epipolarized Light

In conjunction with epipolarized light the gold/silver enhancement also gives a brilliant image of the signal against a dark background in the light microscope. In this arrangement the epipolarized light source (usually UV) is reflected by the perfectly spherical silver 'mirrors' formed by enhancement of the gold particles. See SPI-Mark Gold Probes for Light Microscopy. Sensitivity is increased by 10x in comparison with normal bright field viewing. This means that lower levels of gold are needed for visualisation, thus producing a much improved peak to background signal. Most modern light microscopes can easily and inexpensively be equipped with an epipolarizing light attachment by the manufacturer.

Electron Microscopy (TEM)

Any gold label on an electron microscope specimen (see Gold conjugates for EM) may also be effectively enhanced with the LM/EM Silver Enhancing Kit. Silver enhancing for electron microscopy is especially useful where a low labeling intensity, due to very low levels of tissue antigen, requires that 1nm or 5nm gold conjugates must be used. With these small particles the best possible labeling intensity is achieved but the particles may not be so easily visualized, especially in electron microscopes of limited magnifying power. By simply floating the EM grid carrying the ultrathin specimen on a drop of enhancing solutions for a few minutes the particles become large enough to see at much lower magnifications. It is quite common to observe these particles, which can be grown to 50nm or more, at only 10,000x magnification or lower. The immunolabeling can therefore be observed within a much wider specimen area than at high magnification. The gold particles grow in a perfectly Spherical manner but when adjacent particles touch they fuse to form elliptical shapes. The technique is so simple that specimens may be enhanced repeatedly and observed in the microscope until the required particle size is achieved.

Scanning Electron Microscopy (SEM)

For SEM studies any gold particle can be used in the immunogold reaction and these particles grown to a size (e.g. 50nm) which is convenient to view by scanning using backscattered electrons or X-ray mapping. The use of small gold particles, e.g. 5nm, with subsequent silver enhancing is a preferred approach to labeling SEM specimens compared to the use of larger gold particles for direct viewing because of the higher labeling intensity.

Test Strips

Provided with the kit is a pack of test strips that have been dot blotted with gold conjugate serially diluted to provide a decreasing protein content. There are 5 spots carrying gold particles conjugated to 10ng, 1ng, 100pg, 10pg, and 1pg of protein. The sensitivity of the kit may be tested by adding 5-10 drops of both initiator and enhancer together in the tube (note that the Silver Kit should reach room temperature first and the bottles shaken before use). The spots will change from pink or invisible - yellow - orange - light brown - dark brown - black. All 5 spots will eventually be stained (in about 20-30 minutes). The strips are intended to be used to judge the stability of the kit after long term storage.

LM/EM Silver Enhancement Kit 1 bottle each of Initiator and Developer

	SPI #	Each Kit	In Stock
2 x 15ml	04180-AB	$ 158.00	No
Test Strips (Pk of 10)	04181-AB	35.90	Yes

Storage Conditions: Store under refrigeration
Non-hazardous from stand point of shipping

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