Cryosectioning, details

Cryosectioning. For some antigens in very low titer, the above techniques may not be successful. Cryoultrathin sectioning, developed by Tokuyasu (60), followed by labeling, may be the method of choice. This method is perhaps the most technically complex and difficult of all TEM specimen preparation techniques. A well-frozen sample is a necessity and this method also requires an ultramicrotome with the cryounit attachment. The specifics of this technique and experimental procedures have been discussed in great detail in two reviews (60, 61). 

Figure 2 The effects of specimen preparation on structure and element content of biological tissues. (A) Conventionally prepared section of heart muscle. Individual cells, their nuclei, the mitochondria, the Z-lines, and the myofilaments are clearly seen. (B) A heart muscle cell prepared by cryofixation and cryosectioning. Although the cellular structures are the same as in (A), the details are not as distinct. (C) A spectrum derived from material as in (A). The elements that can be Identified are Cu from the grid and Os, Pb, and U from the stain. The only element that can be detected from the specimen itself is S. (D) A spectrum derived from cryoprepared heart tissue. Here the major inorganic elements - Na, Mg, P, S, Cl, and K - are detected.

Early results with cryomicrotomes were described by Cobbold and Mendelson [80]. Polyurethane elastomer, a blend of crystalline and noncrystalline polymers, showed spherulitic textures after sectioning at about -70°C. Injection molded polypropylene (PP) was also sectioned at about -70°C, while polytetrafluoroethylene (PTFE) was sectioned at much lower temperatures. The authors concluded that the technique, though difficult, had potential. Extruded styrene-butadiene-styrene (SBS) copolymer was prepared by cryosectioning with a diamond knife in liquid air at —85 to —115°C, followed by osmium tetroxide vapor staining for one hour [81]. This method revealed the alternating sequence of the polystyrene and polybutadiene lamellae. Odell et al. [82] prepared extruded triblock copolymer by first chemically hardening the polybutadiene, with osmium tetroxide, followed by cryoultramicrotomy to produce 30 nm thick sections which showed fine structure details. Parallel polystyrene rods were observed in the SBS copolymer. Ultramicrotomy and selective staining with osmium tetroxide was also used in the preparation of a binary blend of PP and thermoplastic rubber [83]. 

Fig. 5.56 A phase contrast optical micrograph (A) of an impact modified nylon shows the fine dispersion of modifier in the matrix. TEM micrographs of a cryosection, stained with ruthenium tetroxide (B and C) show more detail and finely dispersed subinclusions (arrows) within the elastomeric phase. STEM (D) of an imstained cryosection shows less dense regions in a darker background due to mass loss of the rubber phase during exposure to the electron beam, resulting in contrast enhancement.

Most of the published studies have used probes which detect all mRNA isoforms of a given RAR or RXR. It is also possible to generate isoform-specific riboprobes by limiting the template DNA to the specifically spliced region. In situ detection of individual RAR (RXR) isoforms poses problems, however, because of limitation in the nucleotide length of isoform-specific probes (e.g., 400-500 nt in the case of RAR isoforms), and in the very low abundance of individual mRNA isoforms Nevertheless, there have been recent reports of ISH detection of RARP isoforms in chick embryos (17,18) and RARy isoforms can be specifically detected on cryosections of mouse embryos (unpublished data) Detailed procedures for cloning, amplification, and purification of plasmid DNA, which are beyond the scope of this chapter, can be found in refs. 20 and 21... 

Fig. 4.17 STEM micrographs show the nature of the dispersed phase in an unstained thick section of a polymer blend (A). The combination of cryosectioning and ruthenium tetroxide staining results in somewhat more detail in a TEM micrograph (B).

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