Resin-embedded sections

MICROWAVE HEAT-ASSISTED IMMUNOLABELING OF RESIN-EMBEDDED SECTIONS... 

Sormunen, R., and Leong, A. S.-Y. 1998. Microwave-stimulated antigen retrieval for immunohistol-ogy and immunoelectron microscopy of resin-embedded sections. Appl. Immunohistochem. 6 234-237. 

FIGURE 4.3 High magnification transmission electron micrographs of multilamellar membrane structures in the intercellular space of the cornified part of human epidermis. (A) cryo-electron micrograph of vitreous section. (B, C) conventional electron micrographs of resin embedded sections. The cell plasma membranes appear as 3.8 nm wide bilayers in (A) (open white arrow). A 16 nm broad zone of electron dense material, the cornified cell envelope (white asterix), is directly apposed to the cytoplasmic side of the bilayer plasma membranes in the native sample (A) (open white arrow). Scale bar 50 nm (A). Scale bars 25 nm (B, C) adapted from measures given in Swartzendruber et al. (1989). (A) reprinted from Norlen (2003). With permission from Blackwell Science Publications. (B, C) reprinted from Swartzendruber et al. (1989). With permission from Blackwell Science Publications. 

Sections for lightmicroscope radioautography originate either from par-affin/paraplast or, more recently, resin-embedded specimens as mentioned. 

Prior DAM, Oparka KJ, Roberts IM. En bloc optical sectioning of resin-embedded specimens using a confocal laser scanning microscope. JMicrosc 1998 193 20-27. 

Figure 2 shows STEM images of a 8%wt Pd/2%wt Pt catalyst which is supported on charcoal. The sample was prepared for microscopy by embedding in epoxy resin and sectioning with a diamond knife in an ultramicrotome, and was examined in a Vacuum Generator s Ltd HB5 STEM, with a 5A probe. The sample thickness is about 500A. 

If epoxy resin-embedded tissue is used, cut 2- jm-thick sections with a glass knife, mount on APES-coated slides, and dry as described in Note 2. Deplasticize the sections by immersing them in sodium eth(meth)oxide for 15 min (8). Wash the sections twice with equal parts of methanol (or IMS) and xylene, twice with methanol, for 3 min each, and rehydrate. Afterward, the same HIER and immuno-histochemical protocols are employed as in paraffin sections. 

Epoxy resin-embedded semithin sections of 1-2 j,m may also be used following extraction of the resin with a sodium meth(et)hoxide treatment for 5-8 min. Sodium meth(et)hoxide is prepared by saturating methanol or ethanol with sodium hydroxide pellets. 

Heating is effective in antigen retrieval on semithin and thin sections of resin-embedded tissues. This results not only from the breakdown of protein crosslinks introduced by aldehyde but also from the breakage of bonds between the epoxy resin and the embedded tissue (see Fig. 7.2). It is known that epoxy resins form covalent bonds with tissue proteins during embedding. 

Labeling in the microwave oven is usually carried out at 37°C for 15 min. Longer durations and higher temperatures may result in undesirable changes in antibody concentration and molarity of the salts and pH. After heat treatment, the sections should be kept at room temperature for at least 2 min to stabilize the antibody-antigen complexes. The step-by-step procedure for microwave heat-assisted immunolabeling of resin-embedded thin sections for electron microscopy follows (Rangell and Keller, 2000) ... 

However, electron microscopy of conventional resin-embedded ultrathin sections cannot visualize any membranous structure around the LD. In the ultrathin section... 

There are two different ways to perform electron microscopic immunocytochemistry pre-embedding and postembedding (Stirling, 1990). Pre-embedding electron microscopic immunocytochemistry applies the antibodies and label to samples just after fixation but before embedding in epoxy resin and sectioning. Postembedding electron microscopic immunocytochemistry applies antibodies and label to thin sections made after the samples have been embedded in epoxy resin and sectioned. 

Postembedding electron microscopic immunocytochemistry - a method that uses antibodies applied to sections after embedding in epoxy resin and sectioning with an ultramicrotome. 

This technique of drying and resin-embedding provides the best quality thin sections for microscopy. The method (described in Appendix I and illustrated in Figure 7) is adapted after Jim (1985), Polysciences Inc. (1986), Lamoureux (1994) and Pike Kemp (1996) and uses low-viscosity Spurr epoxy resin (Spurr, 1969). Some of the component chemicals of this resin are toxic, and the operation requires a fume cupboard. 

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