8 Plastic embedding for light microscopy
Ultrastructural studies
In the early development of electron microscopy, extremely hard ester waxes were used with limited success. They were unsuitable for ultrastructural studies as they did not offer sufficient support for ultra-thin sections (approximately 30–80 nm), and because they were unable to withstand the high-energy electron beam that passes through the section within the electron microscope (see Chapter 22). The introduction of plastic/resin embedding media during the 1950s provided improved results and stimulated the development of electron microscopy. Nunn (1970) and Glauert (1987) discuss the properties of embedding media suitable for ultrastructural studies.
Plastic embedding media
Epoxy plastics
Various epoxy plastics have found their widest application as embedding media for ultrastructural studies, because the polymerized plastic is sufficiently hard to permit sections as thin as 30–40 nm to be cut, and it is stable in an electron beam. Embedding schedules for the different epoxy resins used in electron microscopy are given in Chapter 22, and only a brief outline of their properties and uses is given here. Epoxy plastics derive their name from the active group through which they polymerize (Fig. 8.1).
Cutting and staining epoxy sections for light microscopy
To an observer experienced in its interpretation, there is little doubt that for high-resolution light microscopy, toluidine blue is the most useful and informative stain applied to tissue sections embedded in an epoxy plastic. If the stain is heated and used at high alkaline pH, it easily penetrates the plastic and stains various tissue components a blue color of differing shades and intensities, with no appreciable staining of the embedding medium. The staining intensity of tissue components by toluidine blue largely reflects its electron density, and the ultrastructural appearances on subsequent electron microscopy can be partly predicted by the appearances at light microscopy level. For those who prefer polychromatic stains, various formulations, e.g. Paragon, can be used which resemble H&E staining. Many staining techniques can be applied after the surface resin has been ‘etched’ using alcoholic sodium hydroxide (Janes 1979), but the results are not always reliable. Another type of pre-treatment consists of oxidizing osmium-fixed tissue without etching (Bourne & St John 1978) so that aqueous solutions can be stained more consistently.
A few reports have described the application of immunohistochemistry to epoxy sections for light microscopy studies following treatment with sodium ethoxide/methoxide (Giddings et al. 1982; McCluggage et al. 1995; Krenacs et al. 2005), but this practice is seldom used. In general, for the majority of occasions when high-resolution light microscopy is required, it is preferable to use acrylic plastic sections because of their potential easier handling and quality of staining achieved, although some techniques such as immunohistochemistry (as discussed later) have presented tough challenges.
Applications of acrylic sections
The development of acrylic plastic embedding media has usually been stimulated by a requirement for a specific application. Most applications are for light microscopy, but as understanding of the formulation of acrylics has increased, so too have various plastics been introduced which may also be useful for some electron microscopy studies. Some of these plastics, such as the Lowicryls, have been developed mainly for electron microscopy alone (Carlemalm et al. 1982; Acetarin et al. 1986), whereas LR White and Unicryl (Scala et al. 1992) can be used for either purpose. However, for various technical reasons, not all dual-purpose plastics are practical for routine high-resolution light microscopy studies.
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