SPI-Mark™ Colloidal Gold Reagents
Fixation
Cells and tissues may be fixed for subsequent examination or may be
sometimes labelled in the unfixed state. Fixatives either denature tissue
proteins by coagulation (e.g. acetone or methanol) or by forming additive cross
linking compounds (e.g. aldehydes), or both. The resulting complexes
inevitably differ from the unfixed protein in both their chemical and antigenic
profiles. Each tissue requires its own fixation protocol. For example, too much
cross linking in a tissue with high protein density may mask many antigens. On
the other hand, a loose tissue with low protein content may disintegrate
without adequate fixation and antigens may simply be washed out.
For many tissues the best compromise is a mixture of formaldehyde (e.g. 2-
4%) for rapid stabilising with low cross linking, and weak glutaraldehyde (e.g.
0.1%) for greater structural preservation. For cytological investigation a
precipitating or coagulating fix such as acetone or methanol may be preferred.
Formal acetone has also been used for fixing cell preparations. In some
studies simple air drying may allow enough antigenic preservation for
immunolabeling.
Post fixation for electron microscopy has mostly involved the use of osmium
tetroxide in order to preserve membrane components and provide image
contrast. The introduction of osmium into tissue is, however, not always
desirable and more recently tannic acid has been suggested as an alternative1.
Whatever method of fixation is selected it must serve the dual function of
retaining the essential structural and antigenic components of the tissue
without introducing any material which may interfere with the labeling. In some
cases the introduction of heavy metals such as osmium or uranium into the
tissue may cause increased non specific labeling and must be treated with
caution.
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Thursday May 17, 2012
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