SPI-Mark™ Colloidal Gold Reagents
Sectioning
Sections should be mounted on nickel or gold grids for EM investigations.
Copper grids introduce unwanted background into the immunolabeling. For
post embedding labeling section thickness is unimportant since the antibodies
do not penetrate the resin sections. Sections of 100nm thickness should be
adequate. While epoxy resin sections float easily and flatten onto the surface
of water, acrylic resin sections require a more careful approach. Flattening is
best achieved by cutting at the right speed with sharp knives, diamond if
possible. They may be mounted on Formvar coated grids or bare grids. The
user should be aware that Formvar and epoxy resin contains high levels of
sulphur, which produces non-specific attraction of gold particles.
It is also important to remember that it is the proteins at the section surface,
which will be exposed to subsequent solutions for labeling, and that internal
protein will not be accessed in resin sections. In this respect it should also be
noted that the proteins on the section surface will be exposed to water, not
buffer, during the sectioning procedure and that this may affect the
subsequent labeling to some degree. This exposure should be kept as short
as possible to avoid denaturing the proteins. For similar reasons sections cut
from resin embedded tissue will not necessarily have an infinite life time when
stored at ambient conditions. Freshly cut sections are likely to be more
reliable. To avoid leaching of proteins in water some investigators have cut
sections onto buffer immediately before incubating them with antibodies.
If a tissue block is sectioned then it may often be observed that sections from
the outer region of the block will have better structural integrity and lower
labeling intensity than sections from the centre of the block. This is related to
the penetration of the fixative and the degree of cross linking and antigen
retention. When comparing results between incubations always use adjacent
tissue sections.
For LM sections embedded in resin the above considerations are also true. For
wax embedded sections, however, the thicker the section the greater the
labeling intensity after dewaxing. With LM sections mounting securely on
glass slides is of great importance to withstand further incubations. Slides
should be first coated with BIOBOND to eliminate variations in glass
composition from different sources and to provide a firm section adhesive
without introducing non-specific background. Lysine should be avoided at all
costs as a tissue adhesive since it attracts gold very strongly. Chrome alum
also produces significant background.
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Thursday May 17, 2012
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