SPI-Mark™ Colloidal Gold Reagents
Selection of protocol for specific types of samples
There is a wide range of different kinds of samples on which one might want to conduct immunogold
staining and electron microscopy so it should not be surprising that a number of different
protocols have now evolved which are more optimum for specific kinds of samples. We have
listed below the most important ones (e.g. those most frequently used):
Post embedding techniques (labeling of microtomed sections):
This is by far the most widely used technique for the labeling of antigens for
TEM examination. Here the tissue is fixed and embedded and incubations performed
on the sections. Only the surface of the section is labeled. Ultrathin cryosections
(some times called "frozen sections") are usually included in this category.
Pre-embedding labeling of tissues and cells:
In this case, tissues (e.g. cell cultures or cells in suspension) are labeled by
incubation, usually after fixation and before embedding. This method is particularly
appropriate for the identification of antigenic sites on membranes of both isolated cells
and micro organisms.
Negative staining:
This is the most suitable method for the identification of antigens on micro-organisms
dried onto a TEM grid.
Replica technique:
Cell surface antigens on cultured cells may be examined with this method.
Cells are fixed and labeled before being replicated for study by TEM. The replication
method is generally either carbon only or platinum/carbon.
Freeze fracture (and maybe etching):
Freeze fracture is suitable for labeling cell membranes and for examination by thin
sections or with replicas. With this technique it is possible to label plasma and
intracellular membranes on isolated cells or within tissues.
Scanning electron microscopy (SEM):
Large specimen areas may be examined by SEM. Specimen surfaces are labeled by
a pre-embedding method and then prepared for SEM examination. Backscattered
electrons provide an image of the gold particles on the specimen surface.
Large gold particles (larger than 20 nm) may be imaged in the SEM with good resolution
while the specimen surface is viewed with secondary electron emission (e.g. the normal
SEM topographical mode). Alternatively, for higher labeling intensity, smaller (e. g. 5
nm) gold conjugates are used and subsequently silver enhanced.
To obtain good results, the sample must be made surface conductive, and the standard method
has been to carbon coat. However carbon coated samples lack the normal "depth" perception
and are not as desirable as a gold coated sample, but which of course could not be used since
such a coating would block out the BSE image. Recently, it has been shown that a 1 or 2 nm
layer of osmium metal, when deposited with an
OPC osmium coater,
will not only make the sample conductive
and without blocking out any of the BSE gold signal, but the image exhibits outstanding depth perception.
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Tuesday February 09, 2010
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