Introduction to microtechnique

1.1. Thickness and contrast 1
1.2. Staining and histochemistry 3
1.3. Some physical considerations 4
1.4. Properties of tissues 5
1.5. Books and journals 6
1.6. On carrying out instructions 6
1.6.1. De-waxing and hydration of paraffin sections 6
1.6.1.1. Solvent method 6
1.6.1.2. Emulsification method 7
1.6.2. Staining 8
1.6.3. Washing and rinsing 8
1.6.4. Dehydration and clearing 9
1.6.5. Staining through paraffin: an irrational method 9
1.6.6. Mechanization 10
1.7. Whole mounts and free cells 10
1.8. Understanding the methods 10

Many theoretical explanations and practical instructions are contained in this book. The present chapter concerns aspects of the making of microscopical preparations that are fundamental to all the techniques described in the later chapters. It cannot be over-emphasized that unless the student or technician understands the rationale of all that is to be done, he will not do it properly. Chapter 4 and Section 1.6 of this chapter contain some of the practical information relevant to the manipulations discussed in all parts of the book.

With an ordinary light microscope (LM) it is possible to see only limited structural detail in a living or freshly removed part of a large organism. For the resolution of finer structure within and around cells it is necessary to study fixed specimens. These are pieces of animal or plant material that have been structurally stabilized, usually by a chemical treatment. Fixation, which is reviewed in Chapter 2, arrests post-mortem decay and also gives a harder consistency to many tissues. Fixation introduces structural and chemical artifacts, but these are fairly well understood and for most purposes they outweigh the technical difficulties and artifacts encountered in the examination of unfixed specimens. Some naturally hard materials require softening treatments after fixation (Chapter 3); bone, for example, can be decalcified.

1.1. Thickness and contrast

In order to be examined with a microscope, a specimen must be thin enough to be transparent and must possess sufficient contrast to permit the resolution of structural detail. Thinness may be an intrinsic property of the object to be examined. Thus, small animals and plants, films or smears of cells, tissue cultures, macerated or teased tissues, and spread-out sheets of epithelium or connective tissue are all thin enough to mount on slides directly. In histology, histopathology and histochemistry, one is more often concerned with the internal structure of larger, solid specimens. These must be cut into thin slices or sections in order to make them suitable for microscopical examination. Methods also exist for examination of surfaces: notably scanning electron microscopy (Echlin, 2009), atomic force microscopy (Vesenka et al., 1995), and in vivo light microscopic examination of mucosal surfaces for clinical diagnosis (Bornhop et al. 1999; Goetz et al. 2013). Preparative methods for such techniques are outside the scope of this book.

Freehand sections, cut with a razor, are rarely used in animal histology but are still sometimes employed for botanical material. Though some expertise is necessary, sectioning in this way has the advantage of requiring little in the way of time or special equipment. In a variant of the freehand technique, single sections of animal tissues are obtained by fixing a specimen with cyanoacrylate glue to either a glass slide or a cellulose acetate sheet, and then shaving it off with an inclined razor blade (Troyer et al., 2002; Dobkin and Troyer, 2003). When sections of human or animal tissues are needed in a hurry, frozen sections are commonly used. The cryostat, a microtome mounted in a freezing cabinet, may be used for cutting thin (5–10 µm) sections of either fixed or unfixed tissue. Each frozen section is collected from the knife onto a glass slide or coverslip, and does not thaw until it is removed from the cryostat cabinet. A traditional freezing microtome, with which the sections thaw as they are being cut, is used when thick (50–100 µm) sections are needed, especially in neuroscience research. Another advantage of cutting frozen sections, aside from speed, is the preservation of lipids, most of which are dissolved out during the course of dehydration and embedding in paraffin or plastic. A vibrating microtome (Vibratome) can cut thick (50–100 µm) sections of unfixed, unfrozen specimens. The blade of this instrument passes with a sawing motion through a block of tissue immersed in an isotonic saline solution. The cutting process is much slower than with other types of microtome, so it is not feasible to prepare large numbers of sections. Vibratome sections of fixed material are similar to sections cut with a freezing microtome, but they do not contain holes or other artifacts associated with ice crystal formation.

When the preservation of lipids or of heat-labile substances such as enzymes is not important, fixed specimens are dehydrated, cleared (which means, in this context, equilibrated with a solvent that is miscible with paraffin), infiltrated with molten paraffin wax, and finally, embedded (blocked out) as the wax cools and solidifies. Paraffin sections are most commonly cut on a rotary microtome, though a rocking microtome or a sledge microtome may also be used. The sections come off the knife in ribbons, and with sufficient skill it is possible to obtain serial sections as little as 4 µm thick through the whole block of tissue. The traditional embedding medium for large specimens was cellulose nitrate. Materials sold for the purpose were commonly called nitrocellulose, celloidin or low-viscosity nitrocellulose (LVN); these embedding media are seldom used today (see Chapter 4, Sections 4.1.3, 4.1.4). Various synthetic resins (plastics) are also used as embedding media for light microscopy, though their main application is in the cutting of extremely thin sections for examination in the electron microscope (EM). Resin-embedded tissue is usually sectioned...