Silver Kits

Silver Enhancing Kit for Light or Electron Microscopy

The BBlnternational Silver Enhancing Kit is a simple to use and sensitive method for enhancement of immunogold labelled specimens to be examined in the light microscope. It depends on the reduction of silver from solution in the presence of a developer by the gold label attached to proteins / antibodies / DNA on cells and tissues mounted on a supporting glass slide or coverslip. It may also be used on monolayers of cells prepared and handled within a culture dish for direct examination in the microscope. The kit has been specially formulated by BBlnternational to combine ease of use with good sensitivity and contrast. The components of the solutions have been selected to allow good control over time of development with minimal background. Because of its relative insensitivity to light the slides may be monitored in normal lighting on the microscope stage. The slides / specimens need no fixing after development, the stain is permanent and the slides may be counterstained in the normal manner. The development operates at pH7.4 to minimise the difficulties experienced with other enhancing methods where very low pH sometimes causes the extraction of labelled proteins from the specimen. A MSDS is available for this product.

Procedure
1. The specimen should be labelled with the appropriate BBI immunogold reagents for light microscope application. Both 1nm and 5nm BBI gold conjugates are recommended for application to LM specimens. The 5nm conjugates give good specific labelling of dewaxed paraffin sections, resin sections and cryostat sections. In addition whole cells and smears may similarly be labelled. See the Conjugate Technical Instruction section. Greater intensity of labelling may be achieved with the BBI 1nm gold conjugates. This is especially so when penetration of cell membranes is required for the labelling of whole cells for example. In addition, the 1nm gold conjugate penetrates further into cryostat and dewaxed sections compared with larger sizes. 2. Wash the specimen extremely thoroughly with deionised or distilled water. Single slides may be handled individually and rinsed continuously for 2 minutes with a jet of water. Slides in batches may be washed in glass containers with several (e.g. 5) changes of water. If required the slides may be stored dried at 4C for several weeks after through washing in water before silver enhancing.

3. Dry the area around the specimen. Allow the Silver kit to reach room temperature first and shake the bottles well before mixing 3 drops of initiator and 3 drops of enhancer together in a clean plastic or glass tube. Apply the solution as a drop onto the specimen. The development may be monitored continuously on the microscope slide, but high intensity illumination should be avoided over a long time. 4. The development of silver deposits at the site of gold labelling is dependent on temperature. At 20C a typical development time is approximately 10-15 minutes for the enhancement of 5nm conjugates. With 1nm gold conjugates a typical enhancing time is 20 minutes. Lower temperatures require a longer development time; higher temperatures require a shorter development time. The solutions have been formulated to allow the user to easily control the time of development. The development should be stopped when specific staining is complete and background is still minimal. The reaction is stopped by washing immediately in tap water for 1-2 minutes. If performing enhancement of whole cells labelled intracellularly, more extensive washing may be required to remove all traces of developer from the cells. No fixing is required. A colour change will be seen as the reduction of silver from solution occurs on the specimen. The specifically labelled sites will change from invisible - light brown -dark brown - black. If required the procedure may be continued by using freshly mixed solutions. Counterstain the specimen as normal. Mount and view in the light microscope.

Test strips
Provided with this kit is a pack of test strips that have been dot blotted with gold conjugate serially diluted to provide decreasing protein content. There are five spots carrying gold particles conjugates 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 and the bottles shaken beforeuse). The spots will change from pink or invisible - yellow - orange - light brown - dark brown black. All five 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. Further packs of test strips are available from BBlnternational. Pack of 10 strips Catalogue Number SETS10.

Storage
Although the intensification kit is relatively insensitive to light during the time of incubation, it should be stored at 4C and in the dark. When properly stored the reagents are stable until their use by date. Shelf life may be extended by storing at 20C. We recommend testing the activity of a kit that has been stored for a period of time without use by using the test strip provided.

Silver Enhancing Kit Blotting

The BBInternational Silver Enhancing Kit is a simple to use and sensitive method for enhancement of immunogold labelled antigens on a solid support. It depends on the reduction of sliver from solution in the presence of a developer by the gold label attached to proteins / antibodies / nucleic acids mounted on a solid phase for direct viewing by eye. Although the intensification kit is relatively insensitive to light during the time of incubation, it should be stored at 4C and in the dark. The kit has been specially formulated by BBInternational to combine ease of use with good sensitivity and contrast. The components of the solutions have been selected to allow good control over the time of development with minimal background. Because of its relative insensitivity to light the enhancement may be monitored in normal room lighting during development. The specimens need no fixing after development but should be thoroughly washed in tap water to remove any traces of the silver solutions. The development operates at pH7.4 to minimise the difficulties experienced with other enhancing methods where very low pH sometimes causes the extraction of labelled proteins from the specimen. An MSDS is available for this product.

Procedure
1. The specimens should be labelled with the appropriate BBI immunogold reagents for biochemical application. Both 1nm and 20nm BBI gold conjugates are recommended for application to antigens immobilised on a solid phase support. 20nm gold conjugates offer the advantage of greater visibility of the gold label before enhancement with silver. Greater intensity of labelling may be achieved with the BBI 1nm gold conjugates due to the larger number of gold particles attaching to the antigen. See the BBI Technical Instruction Booklet describing gold labelling procedures and provided with these products. 2. After labelling with BBI gold conjugates the solid phase support should be thoroughly washed in deionised or distilled water. In all cases non-metallic labware should be employed. Avoid the use of metallic holders and forceps since metals will cause silver precipitation out of solution. A plastic tray is ideal for incubations. 3. Allow the Silver Kit to reach room temperature first - shake the bottles well before use. Mix equal quantities of initiator and enhancer together, enough to cover the surface of the solid phase support to a depth of 3-4mm when in the plastic tray. The development may be monitored in normal room lighting

conditions. Avoid performing the reaction near to strong lighting or in direct sunlight. 4. The development of a silver deposit at the site of gold labelling is dependent on temperature. At 20C typical development time is approximately 15-20 minutes for the enhancement of 20nm gold conjugates. With 1nm gold conjugates a typical enhancing time is 30-40 minutes. Lower temperatures take longer; higher temperatures require a shorter time. The solutions have been formulated to allow the user to easily control the time of development. The development should be stopped when specific staining is complete and background is still minimal. The reaction is stopped by washing immediately in several changes of tap water for 2-3 minutes. No fixing is required. A colour change will be seen as the reduction of silver from solution occurs on the solid phase support. The specifically labelled sites will change from invisible - yellow - orange -light brown - dark brown - black. If required, the procedure may be continued by using freshly mixed solutions. After thorough washing in water allow the support to dry in air.

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. Further packs of test strips are available from BBInternational. Pack of 10 strips Catalogue Number SETS10.

Storage
Solutions should be stored at 4C when not in use. If the kits are not going to be used for long periods of time, it is possible to prolong their life by storing at -20C. If a frozen kit passes its use by date, it may still be possible to use the kit, but it is advisable to test it first using the strip provided.