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Silver Enhancing Kit-Light Microscopy (*)
CAT# RDI-SEKL15, new cat#: 55R-SEKL15 Price: $209.00/kit
The 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.
Although the intensification kit is relatively insensitive to light during the
time of incubation, it should be stored at 4C or frozen and in the dark over
the long term. When properly stored the reagents are stable for at least 12
months at 4C and indefinitely when frozen.
STORE AT 4C OR FROZEN AND AWAY FROM EXCESSIVE HEAT AND LIGHT.
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 time of development with minimal background. Because of
its relative insensitivity to light the slides may be monitored in normal room
lighting on the microscope stage. The slides/specimens need no fixing after
development but may simply be thoroughly washed in tap water. If properly washed after development, the stain is
permanent and the slides may be counterstained in the normal manner. The developement
operates at pH7.4 to minimise the difficulties extraction of labelled proteins
from the speciment.
If contact with the solutions is made by the skin simply wash the area under
tap water.
Procedure
1. The specimens should be labelled with the appropriate immuno gold reagents for light microscope application. Both 1nm and 5nm gold conjugates are recommended for application to LM specimens. The 5nm conjugates give good specific labelling of dewaxed paraffin sections, resin sections and cryostat sec tions. In addition whole cells and smears may similarly be labelled. See the booklet provided with the products for the use of gold reagents. Greater intensity of labelling may be achieved with 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 conjug ate penetrates further into cryostat and dewaxed sections compared with the larger sizes.
2.
3. Dry the area around the speciment. Mix 3 drops of initiator and 3 drops of
enhancer together in a clean plastic/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 deposit 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 gold conjugates. With 1nm gold conjugates a
typical enhancing time is 20 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 simply 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 color 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 simply using freshly mixed solutions.
5. 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 a de-reasing 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 simply by adding 5-10 drops of both initiator and enhancer together in the tube. 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 availabe. Pack of 10 strips -CAT # SETS10
For In vitro Research Use Only
RDI Division of Fitzgerald Industries Intl
phone (978) 371-6446 or (800) 370-2222
fax (978) 371-2266
EMAIL:antibodies@fitzgerald-fii.com