Resin embedding freeze-substitution

Murray, G. J. (1992) Enzyme histochemistry and immunohistochemistry with fteeze-dned or freeze-substituted resin embedded tissue. Histochem. J. 24,399-408... 

The state of the art in fine-structure preservation for thin sectioning can be achieved by using fast-freezing technology followed by freeze-substitution and embedding in resin. The reason that fast freezing is so much better has to do with the speed of fixation, which... 

Once frozen, tissues can be processed in a variety of ways before viewing in the EM, but in this chapter, we only discuss freeze-substitution and embedding in resin (Howard and O Donnell 1987 Steinbrecht and Muller 1987). Resin-embedded samples lend themselves to thin sectioning, which is the most familiar way of viewing cell fine structure for morphology and immunolabeling. 

Lauchli, A., Spurr, A.R. Wittkop, R.W. (1970). Electron probe analysis of freeze substituted, epoxy resin embedded tissue for ion transport studies in plants. Planta, 95, 341-50. 

In a previous review of this topic (5), it was suggested that molecular distillation drying followed by resin embedding needed more work before it could be evaluated as a preparative technique for microanaly-tical work as there had only been one investigation that had used it (59). Unfortunately, as far as this author is aware, there has only been one more study (12) that has used the technique to prepare material for micro-analysis. It is possible that workers have been put off using it by the apparent fall from grace of the related freeze substitution technique or, more likely, by the expense of molecular distillation drying apparatus. 

Fixation by rapid freezing followed by either freeze substitution or cryosectioning can also overcome some of the problems of standard immersion fixation and resin embedding. These are more specialized techniques and will not be dealt with here. Discussion of the methods can be found in Polak and Varndell (6), Hayat (7), and Verkleij and Leunissen (8). 

Fig. 3 shows SFM image of one of the sections (thickness of - 100 nm) of K562 cell embedded in epoxy resin. It should be noticed that topographical contrast and the identification of the K562 internal ultrastructure critically depend on the procedure of cell preparation before embedding (chemical fixation or high-pressure freezing and freeze-substitution). 

Freeze-substitution is perhaps of more use here the sample is frozen rapidly and then held in an organic solvent such as ethanol for several days at — 80°C to remove the ice. After embedding in a resin and polymerization with a UV source, sections are cut and, if necessary, stained. The resultant section will be ideal for any examination and most types of analysis. 

The alternatives to preparing and labelling thawed cryosections are the two resin embedding methods PLT and freeze-substitution. The simplest to do is the PLT method which can be completed without the necessity of expensive equipment. 

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