Fixation, usually by a chemical or mixture of chemicals, permanently preserves the tissue structure for subsequent treatments. Specimens should be immersed in fixative immediately after they are removed from the body. Fixation is used to:  terminate cell metabolism, prevent enzymatic degradation of cells and tissues by autolysis (self-digestion), kill pathogenic microorganisms such as bacteria, fungi, and viruses, and  harden the tissue as a result of either cross-linking or dena turing protein molecules.
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 (most often cross-linked lysine residues). Because formaldehyde does not significantly alter their three-dimensional structure, proteins maintain their ability to react with specific antibodies. This property is important in immunocytochemical staining methods (see page 7). The standard commercial solution of formaldehyde buffered with phosphates (pH 7) acts relatively slowly but penetrates the tissue well. However, because it does not react with lipids, it is a poor fixative of cell membranes.
In the second step, the specimen is prepared for embedding in paraffin to permit sectioning.
Preparing a specimen for examination requires its infiltration with an embedding medium that allows it to be thinly sliced, typically in the range of 5 to 15 m (1 micrometer [ m] equals 1/1,000 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 as high as 100% alcohol to remove water. In the next step, clearing, organic solvents such as xylol or toluol, which are miscible in both alcohol and paraffin, are used to remove the alcohol before infiltration of the specimen with melted paraffin.
TABLE Commonly Used Linear Equivalents1.1 1 picometer (pm) 0.01 angstrom (Å) 1 angstrom 0.1 nanometer (nm) 10 angstroms 1.0 nanometer 1 nanometer 1,000 picometers 1,000 nanometers 1.0 micrometer ( m) 1,000 micrometers 1.0 millimeter (mm)
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 using mounting medium (pinene or acrylic resins) as an adhesive.
In the third step, the specimen is stained to permit examination.
Because 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. Figure 1.1 shows the results of staining with hematoxylin alone, eosin alone, and hematoxylin with counterstain eosin. 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.
Formalin does not preserve all cell and tissue components.