Boxes

box i.i. Functional Considerations: Feulgen Microspectrophotometry 8

box 1.2. Functional Considerations: Development of Electron Microscopy 14

box 1.3. Functional Considerations: Special Staining for Transmission Electron Microscopy 15

box 1.4. Proper Use of the Light Microscope 16

s7 OVERVIEW OF METHODS USED IN HISTOLOGY

The objective of a histology course is to lead the student to understand the microanatomy of cells, tissues, and organs and to correlate structure with function

The methods used by histologists are extremely diverse. Much of the histology course content can be framed in terms of light microscopy, which students use in the laboratory ex ercises. More detailed interpretation of microanatomy rests with the electron microscope (EM), both the transmission electron microscope (TEM) and the scanning electron microscope (SEM). The EM, because of its greater resolution and useful magnification, is often the last step in data acquisition from many auxiliary techniques of cell and molecular biology. These auxiliary techniques include

• Histochemistry and cytochemistry

• Autoradiography

• Organ and tissue culture

• Cell and organelle separation by differential centrifugation

• Specialized microscopic techniques and microscopes

The student may feel removed from such techniques and experimental procedures because direct experience with them is usually not available in current curricula. Nevertheless, it is important to know something about specialized procedures and the data they yield. This chapter provides a survey of methods and offers an explanation of how the data provided by these methods can help the student acquire a sound appreciation of histology.

One problem faced by the student in histology is understanding the nature of the two-dimensional image of a histologic slide or an electron micrograph and how it relates to the three-dimensional structure from which it came. To bridge this conceptual gap, we must first present a brief description of the methods by which slides and electron microscopic specimens are produced.

S/ TISSUE PREPARATION

Hematoxylin and Eosin Staining With Formalin Fixation

The routinely prepared hematoxylin and eosin-stained section is the specimen most commonly studied

The slide set given each student to study with the light microscope consists mostly of formalin-fixed, paraffin-embedded, hematoxylin and eosin (H&E)-stained specimens. Nearly all of the light micrographs in the Atlas section of this book are of slides from actual student sets. Also, most photomicrographs used to illustrate tissues and organs in histology lectures and conferences are taken from such slides. Other techniques are sometimes used to demonstrate specific cell and tissue components; several of these are described below.

The first step in preparation of a tissue or organ sample is fixation to preserve structure

Fixation, usually by a chemical or mixture of chemicals, stops cell metabolism and preserves the tissue structure for subsequent treatments. Formalin, a 37% aqueous solution of formaldehyde, at various dilutions and in combination with other chemicals and buffers, is the most commonly used fixative. Formaldehyde preserves the general structure of the cell and extracellular components by reacting with the amino groups of proteins; formaldehyde does not react with lipids and, therefore, is a poor fixative of membranes.

In the second step, the specimen is prepared for embedding in paraffin to permit sectioning

To allow the specimen to be examined, it must be infiltrated with an embedding medium that allows it to be thinly sliced, 5 to 15 /xm (1 micrometer l/xm] equals 1/1,000th of a millimeter [mm]; see Table 1.1). The specimen is washed after fixation and dehydrated in a series of alcohol solutions of ascending concentration up to 100% alcohol to remove water. Organic solvents such as xylol or toluol, which are miscible in both alcohol and paraffin, are then used to remove the alcohol prior to infiltration of the specimen with melted paraffin.

When the melted paraffin is cool and hardened, it is trimmed into an appropriately sized block. The block is then mounted in a specially designed slicing machine, a microtome, and cut with a steel knife. The resulting sections are then mounted on glass slides, with albumin used as an adhesive.

In the third step, the specimen is stained to permit examination

Because the paraffin sections are colorless, the specimen is not yet suitable for light microscopic examination. To color or stain the tissue sections, the paraffin must be dissolved out, again with xylol or toluol, and the slide must then be rehydrated through a series of solutions of descending alcohol concentration. The tissue on the slides is then stained with hematoxylin in water. Because the counterstain, eosin, is more soluble in alcohol than in water, the specimen is again dehydrated through a series of alcohol solutions of ascending concentration and stained with eosin in alcohol. The results of staining with hematoxylin alone, eosin alone, and hematoxylin with counterstain eosin are shown in Figure 1.1. After staining, the specimen is then passed through xylol or toluol to a nonaqueous mounting medium and covered with a coverslip to obtain a permanent preparation. A summary of H&E staining reactions of various cell and tissue components is presented in Table 1.2.

Other Fixatives

Formalin does not preserve all cell and tissue components

Although FI&E-stained sections of formalin-fixed specimens are convenient to use because they adequately display general structural features, they are not specific to elucidate the chemical composition of cell components. Also, many components are lost in the preparation of the specimen. To retain these components and structures, other fixation methods must be used. These methods are table i.i. Commonly Used Linear Equivalents

10 Angstroms = 1.0 nanometer (formerly millimicron [m/x])

1,000 nanometers = 1.0 micrometer (/xin) (formerly micron [yu.])

1,000 micrometers = 1.0 millimeter (mm)

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