SPI-Mark™ Silver Enhancement Kit
Use Instructions and Product Information
Silver Enhancing Kit for Light or Electron Microscopy
The SPI-Mark™ Silver Enhancement Kit is a simple to use and sensitive
method for enhancement of immunogold labeled 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 SPI-Mark kit combines 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 a pH of 7.4 to
minimize the difficulties experienced with other enhancing methods where very
low pH sometimes causes the extraction of labeled proteins from the specimen.
Procedure
1. The specimen should be labeled with the appropriate SPI-Mark immunogold
reagents for light microscope application. Both 1nm and 5nm SPI-Mark gold
conjugates are recommended for application to LM specimens. The 5nm
conjugates give good specific labeling of dewaxed paraffin sections, resin
sections and cryostat sections. In addition whole cells and smears may
similarly be labeled. Greater intensity of labeling may be achieved with the
SPI-Mark 1nm gold conjugates. This is especially so when penetration of cell
membranes is required for the labeling 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 demonized 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
4°C 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 labeling is dependent
on temperature. At 20°C 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 labeled intracellularly, more extensive washing may be required to
remove all traces of developer from the cells. No fixing is required. A color
change will be seen as the reduction of silver from solution occurs on the
specimen. The specifically labeled 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 before use). 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 SPI Supplies.
Storage
Although the intensification kit is relatively insensitive to light during the time of
incubation, it should be stored at 4°C and in the dark. When properly stored
the reagents are stable until their use by date. Shelf life may be extended by
storing at –20°C. 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 SPI-Mark Silver Enhancing Kit is a simple to use and sensitive
method for enhancement of immunogold labeled 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 4°C and in the dark.
The kit has been specially formulated 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 minimize the difficulties experienced with other enhancing methods
where very low pH sometimes causes the extraction of labeled proteins from
the specimen.
Procedure
1. The specimens should be labeled with the appropriate
SPI-Mark immunogold reagents
for biochemical application. Both 1nm and 20nm SPI-Mark gold conjugates
are recommended for application to antigens immobilized 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 labeling may be
achieved with the SPI-Mark 1nm gold conjugates due to the larger number of gold
particles attaching to the antigen.
2. After labeling with SPI-Mark gold conjugates
the solid phase support should be
thoroughly washed in deionized 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. We recommend either the
PTFE coated or gold plated tweezers offered by SPI Supplies.
We would add a note of caution at this point: You must use gold plated tweezers as
offered by SPI Supplies and not what some sources call "gold plated" but what they offer
is really a gold "flash", a much thinner coating and one that does not provide for the
protection as one would have from the thicker gold plated tweezers.
3. Allow the SPI-Mark Silver Enhancement 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 labeling is dependent
on temperature. At 20°C 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 color change will be seen as the reduction of silver from solution occurs on
the solid phase support. The specifically labeled 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 SPI Supplies.
Storage
Solutions should be stored at 4°C 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
-20°C. 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
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Sunday March 21, 2010
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