Biomedical Engineering Reference
In-Depth Information
Ultrathin sectioning and plastic embedding
This is one of the most frequently used methods for examining bio-
logical samples. Fixed samples are stained with heavy metals, usually
osmium tetroxide and uranyl acetate, dehydrated with ethanol or ace-
tone to remove water, and embedded in plastic for sectioning. Because
harsh chemicals are used prior to embedding, antigenic structures are
often not retained, and therefore antibodies cannot be combined with
plastic embedding procedures.
Immunogold electron microscopy
To detect antigenic structures within cells and tissues using TEM,
samples are more carefully protected prior to sectioning. The samples
are cryo-protected with agents such as sucrose and flash frozen in liq-
uid nitrogen, which protects the organization of the subcellular struc-
tures. Cryosectioning is then carried out at temperatures as low as
- 120°C using an ultramicrotome. Ultramicrotomes are generally fitted
with diamond-edged knives and are able to cut sections as thin as 60 nm.
Antibodies that are labeled with colloidal gold particles (see Chapter 4)
can be added in order to specifically identify the localization of protein
antigens found within intracellular compartments. Contrast is provided
by staining sections with agents such as uranyl acetate, dried and ex-
amined by TEM. Figure 10 shows an immunogold labeled-section of a
plasma cell that is labeled with an antibody against the IgM antibody
that is being synthesized and secreted by the cell. The section shown
demonstrates the presence of high concentrations of the newly syn-
thesized IgM antibody (denoted by the electron dense colloidal gold on
the anti-IgM antibody) within the endoplasmic reticulum of the cell (the
narrow, membrane contained organelle).
Figure 10. Immunogoldelectron microscopy
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