Appendix

General Principles of Tissue Preparation and Staining

Introduction

The study of tissue structure relies on the preparation of tissue samples in ways that allow their structural details to be viewed at light or electron microscopic levels. First, there is the method of studying tissue using the light microscope. Second, there is the study of what is sometimes called the “ultrastructure” of tissue using the transmission electron microscope. There are numerous variations on these two general themes. Recognizing that a survey of all tissue preparation techniques is well beyond the scope of this atlas, the general features of the two methods mentioned are briefly summarized here.

Preservation versus Fixation

Preservation and fixation of tissues, for subsequent treatments, share to varying degrees the common goals of minimizing any further tissue degradation and preserving the various components of the tissue in as lifelike a condition as possible. While the terms preservation and fixation are frequently used as interchangeable, they are, in fact, slightly different.

Preservation of tissue accomplishes the first goal stated above (prevents further degradation) but not necessarily the second. For example, a can of green peas contains preservatives; these may even be listed on the label. They protect the peas from further degradation. If you take a pea out of the can and look at it microscopically, its structure might be recognizable, but it is not very lifelike. Fish that are preserved in salt undergo significant changes that render the tissue useless for microscopic examination. Such structures are preserved but are not fixed. Many of the substances used to preserve tissues (animal and plant) can be ingested in moderation and cause absolutely no harm.

Fixation, on the other hand, protects against further degradation while preserving the internal components of the cell in a strikingly lifelike appearance. Fixation also hardens the tissue, making it possible to further manipulate the sample without damage. Tissue that is fixed will appear quite lifelike when viewed with a microscope. In this respect, a sample of tissue that is fixed is also preserved; however, a sample of tissue that is preserved is not necessarily fixed. Another important difference is the fact that most substances (except alcohol) used to fix tissue generally cannot be ingested; to do so, even in very moderate amounts, would cause significant harm or death.

Fixatives and Methods of Fixation

Fixatives

In addition to preventing tissue degradation, preserving components of the tissue, and hardening the tissue, proper fixation will also transform the contents of the cell from a semifluid to a semisolid and prepare the cell contents for visualization with stains, dyes, or metallic salts. There are numerous fixatives, many of which are designed for unique applications, which can be used separately or in combinations. Only representative examples are mentioned here.

Formalin (mixture of formaldehyde and alcohol), in solution of 5% to 10%, is one of the more commonly used general fixatives. It penetrates the tissue rapidly, leaves no residues, and requires little or no washing of the tissue to remove. It is used alone, in a solution buffered with sodium phosphate salts, or in other combinations. Buffered neutral formalin is usually preferred. Formalin acts through cross-linking between proteins.

Picric acid, in combination with formalin and glacial acetic acid (Bouin solution), is also used as a fixative. Its action of fixation is not fully understood, and it does not harden the tissue as much as formalin. Details of the nucleus are well demonstrated. However, picric acid in its dry form must be handled with care because it is an explosion hazard. Picric acid can also be used as a stain.

Aldehydes, such as glutaraldehyde and paraformaldehyde, are excellent fixatives for light microscopic applications and are also widely used in electron microscopy. Aldehydes act rapidly but tend to penetrate the tissue slowly. However, they provide excellent cellular detail regarding the contents of the

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cytoplasm and the nucleus. Osmium tetroxide, an oxidizing agent, is also used as a fixative for electron microscopy.

Alcohols, either methyl or ethyl alcohol, are also used as fixatives. Because they cause the tissue to become quite hard and, therefore, brittle, they are usually used in special applications such as smears of tissue or blood. Alcohol in various dilutions is also commonly used in tissue processing.

Mercuric chloride is used in combination with other substances in a variety of fixatives. Though the method of action at the cellular level is not well understood and fixatives containing this substance may penetrate the tissue slowly, they do provide excellent cellular detail.

Acetic acid, though rarely used as a fixative on its own, is used in combination with other agents in a variety of fixatives such as Bouin, Carnoy, and Clarke solutions. Although acetic acid may be used in concentrated form in certain applications, it is primarily used in combinations with other fixatives (and is thereby diluted); it is also commonly used by itself in dilutions of 1% to 5%.

Methods of Fixation

Fixation is usually accomplished in one of two ways. The first is by immersion fixation. The fixative is prepared and a small piece of tissue removed and immersed in the fixative. It is common to suspend the tissue sample in the fixative; one method is to drape a delicate piece of cheesecloth in the fixative and place the sample in the sling thus created. This is advantageous because the fixative can penetrate from all sides of the tissue block. This method is useful when the tissue sample is small and the fixative penetrates rapidly.

The second is perfusion fixation. In this method the fixative is perfused through the intact vascular system of the organism. The fixative is preceded by a wash of physiological saline containing heparin and a substance that will paralyze the vessel walls, such as procaine hydrochloride, to ensure that the vascular bed will not contract in response to the fixative solution. This wash is followed immediately by the fixative. After perfusion, the tissue samples are removed and placed in more of the same fixative used in the perfusion. This method provides superior fixation of large pieces of tissue and is commonly used in many research applications.

FREEZING is also used as a method of fixation, especially in the clinical setting when a rapid diagnosis is needed during a medical procedure. A fresh tissue sample is retrieved from the patient or organism and immersed in liquid carbon dioxide or in a substance (such as isopentane) cooled extremely rapidly by dry ice. The best results are obtained with small tissue samples that are rapidly cooled to very low temperatures (–40° to –60° C). In general, rapid cooling causes very small ice crystals and minimal tissue disruption; slow cooling (like in your freezer) causes large ice crystals and maximum tissue disruption.

Processing of Fixed Tissue

Once the tissue sample is satisfactorily fixed, it must be taken through a series of steps that result in a thin slice of tissue mounted on a glass slide (for light microscopy) or an even thinner slice mounted on a copper grid (for electron microscopy). In general, this process requires three basic steps: (1) The water in the tissue needs to be removed, (2) the tissue must be passed through a solution that is miscible with water and with the substance in which the tissue will be embedded, and (3) the tissue must be passed through the embedding medium. Because the specific details of these steps may relate to the type of tissue being processed, only general comments are made here.

Dehydration: The goal of dehydration is to remove the water from the tissue. The most common method is to start with alcohol in a concentration of 75% to 80%. Recall that alcohol will mix with water; therefore, it can be used to remove water from the tissue. The tissue sample is passed, stepwise, through progressively higher concentrations (from 75%–80% up to 100%) of alcohol; usually more than one step is used at 95% and 100%. Through this process, the water is completely removed from the tissue and replaced with alcohol.

Clearing: The clearing reagent is a substance that will mix both with alcohol and with the embedding medium. The most commonly used are xylene, toluene, and chloroform. The tissue is passed from 100% alcohol through changes of the clearing reagent. This stepwise process progressively removes the alcohol from the tissue and replaces it with the clearing reagent. This reagent is miscible with the embedding medium.

Impregnation and embedding: Because the clearing reagent will mix with the embedding medium, the tissue sample is taken from the last step in this reagent and placed in melted embedding medium (such as paraplast or bioloid—both are types of wax). The sample is then progressively passed through several changes of the embedding material. This stepwise process progressively removes the clearing reagent and replaces it with the embedding medium that will harden when cooled. Embedding is generally done in two steps. First, the tissue is removed from the last impregnation step and immediately placed in melted medium in a vacuum oven. The last traces of the clearing reagent and any minute

bubbles are removed by vacuum. Second, the tissue sample is oriented in an embedding mold. The mold is then filled with melted medium and allowed to cool so the medium hardens. A similar set of steps are followed when a polymer is used as the embedding media for electron microscopy.

A comment on frozen sections: As noted above, rapid freezing can be used to fix tissue. Freezing can also be used to prepare tissues for sectioning. Small pieces can be rapidly frozen, mounted on a microtome, and sectioned. Larger pieces can be immersed in a sucrose solution until fully impregnated with the sucrose (the tissue initially floats and then sinks). The sucrose expedites the freezing process and renders minute ice crystals. The tissue is then mounted, frozen, and sectioned.

Sectioning and Mounting

Sectioning of prepared tissues is done on microtomes that are designed to accommodate sections mounted in paraplast (paraffin), frozen in ice, or embedded in plastic. The sections are cut using extremely sharp metal or glass knives, removed from the edge of the knife either as individual sections or as ribbons of sections, and floated in water (usually warmed) or in other fluids that are required by the unique features of the specific technique. For most applications in light microscopy, the sections range in thickness from about 5 to 12 mm (for paraffin embedded tissue) and from about 0.5 to 2.0 mm (for plastic embedded tissue). Special techniques may require sections up to 40 to 70 mm in thickness. At the opposite extreme of the microscopic continuum, glass or diamond knives are used to cut extremely thin sections for electron microscopy (EM, commonly called “TEM”). The thickness of sections for electron microscopy/transmission electron microscopy (EM/TEM) usually ranges from about 80 to 110 nm (10−9 m).

After the sections are cut, and before they are mounted, it is routine to make sure the glass slides are clean and to treat the slides so that the sections will not come off during the staining process. This may be accomplished by either spreading a drop of albumin on the slide or by adding a small amount of albumin (or gelatin) to the warm water bath in which the tissues are floated. Celloidin can also be used as an adhesive.

Staining

The goal of staining tissue slices is to use substances to impart color to various components of the section, making these components available for study. In many cases, the stain will visualize a specific component, such as fat, neurofibrils, or glycogen.

Stains are large molecules that are characterized by groups that absorb visible light (wavelengths between 380 and 760 nm) and groups that permit the attachment of the stain to the various chemical elements of the cell. The component of the stain that is responsible for light absorption is a system of conjugated double bonds known as the chromophore. The “combining” portion of the stain, which may also function as a solubilizing agent, is the auxochrome.

The simplest way that stains function is to exploit the electrostatic interactions between the stain molecules and components of the cell; positive charges on cellular structures attract negatively charged stain molecules and vice versa. Basic dyes carry positive charges and are, consequently, known as cationic dyes; they are attracted to negative charges within the tissue. Hematoxylin and toluidine blue are commonly used basic (cationic) dyes. They stain nuclear DNA, cytoplasmic RNA, sulfonated polysaccharides such as chondroitin sulfate, and polycarboxylic acids such as hyaluronic acid. Acidic dyes carry negative charges and are, consequently, known as anionic dyes; they are attracted to positive charges within the tissue. Eosin Y is a commonly used acidic (anionic) dye. It stains many proteins (and, therefore, stains many structures within the cell), and acid dyes also stain extracellular structures such as collagen.

There are literally hundreds of substances that are used as stains or dyes, or that may be used to impregnate tissues, and there are equally numerous methods or techniques that use these substances, in various combinations, to visualize the components of cells. Recognizing that even a brief survey of the broad range of methods is well beyond the scope of this atlas; the following focuses on the major stains used in this book.

Hematoxylin and eosin (H&E) is, by far, the most commonly used combination stain in basic sciences for general histological preparations and in clinical medicine for pathological specimens. This combination of cationic and anionic dyes results in most constituents of the cell (RNA, DNA, polysaccharides, and others) being stained various tones of either blue or pink. This method also stains extracellular collagen.

The trichrome stains are usually mixtures of acidic dyes, each having its own ionization constant. The Mallory trichrome stain, which is one of the more common, is a mixture of dyes used to demonstrate connective tissue (collagen stains blue) and other cellular constituents (nuclei and cytoplasm generally stain red), and blood cells (erythrocytes stain yellow). Trichrome stains are particularly

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useful for endocrine organs in which cells that produce different secretions can be stained different colors.

Wright stain is also a mixture of stains containing one acidic dye (methylene blue) and one basic dye (eosin). Wright stain is widely used for blood and bone marrow smears. Basophilic granules are deep purple, acidophilic (or eosinophilic) granules are reddish, and the cytoplasm is usually bluish gray, although it may appear pink to red (or mottled) if it contains acidophilic substances.

Elastic fiber stains are individual or combinations of dyes that stain cellular constituents but will also prominently stain elastic fibers. These exploit relatively nonspecific ionic and nonionic differences between collagen and elastin. Depending on the procedure used, elastic fibers may appear brown, deep purple, blue-black, or black.

Reticular fiber stains, as is the case for elastic stains, also exploit the relatively nonspecific differences in the amount of glycosylation of reticulin versus that of collagen. In general, reticular fibers are stained black, using silver as the chromophore, with the other cellular elements appearing as a monochromatic background of gray or various shades of light red. Whereas reticular fibers stand out in these special stains, other cellular detail does not.

Silver stains represent a large category of what are actually silver impregnation methods, not stains. In general, these methods use dilute solutions of silver nitrate (gold and mercury are alse sometimes used) to impregnate blocks of nerve tissue and precipitate the metal ions on the cell membranes of neurons and glia. Tissue blocks, generally no more than 0.3 to 0.6 cm thick, are fixed in formalin, treated with a mordant solution such as potassium dichromate, suspended in a dilute silver nitrate solution for several days (with block cleaning and solution changing every day), and then encased in paraffin (not embedded). Sections are cut at thicknesses ranging from 40 to 70 mm and mounted on slides. Individual neurons and glia cells appear black on a light-yellow to off-white background. The mechanism of attachment of the silver ions to the cell membranes is not well understood.

The periodic acid-Schiff (PAS) reaction is a method that is more specific in its staining reaction than the anionic, cationic, or lipid soluble dyes. One such stain is the Schiff reagent (leucobasic fuchsin), which reacts specifically with free aldehydes. Pretreatment with periodic acid converts adjacent hydroxyl groups, such as those found in glycogen, into aldehydes. Treatment with the Schiff reagent stains the free aldehyde groups red; the sections are usually counterstained with H&E. The PAS reaction demonstrates sites of high concentrations of polysaccharide-containing components, such as glycogen and glycosaminoglycans.

Immunocytochemistry is a specialized method that can be used to precisely localize enzymes or large molecules (macromolecules) within the cell or on its membrane. The immune system of the body is able to defend itself against foreign molecules (antigens) by producing specific types of proteins (antibodies). Immunocytochemistry methods use this feature of cells to visualize specific molecules. For light microscopy, an antibody is produced (for example, in rabbit tissue) against a specified protein or molecule, and the antibody is coupled with a dye, such as fluorescein or rhodamine B. When this labeled antibody attaches to a specific antigen and is exposed to ultraviolet light, it will fluoresce greenish yellow (fluorescein) or bright red (rhodamine B), thereby specifically identifying the location of that molecule. This is the direct method: An antibody is produced, coupled to a dye, attached to an antigen, and, thus, becomes visible. In the indirect method, unlabeled antibodies are produced in one animal (rabbit) against a specific antigen and then applied to a tissue to which they attach. The unlabeled antibodies are visualized by exposing them to labeled antibodies that are made in another species (goat) and that are directed against the immunoglobulins from the first species. One way to visualize this is as follows: An antibody is produced, not labeled, and attached to an antigen; a second antibody is produced, coupled with a dye, attached to the first unlabeled antibody, and, thus, then becomes visible.

Transmission and Scanning Electron

Microscopy

In slightly over 60 years, TEM has gone from a scientific curiosity to an essential tool in the study of tissue structure. The general steps used in TEM are conceptually similar to those used in light microscopy but customized to meet the unique needs of this technique. The tissue blocks are quite small, usually about 1 cubic millimeter, specially fixed to retain the fine integrity of the cellular constituents, and infiltrated with unpolymerized plastic rather than with wax or fluid. Sections usually in the range of 0.02 to 0.1 mm are cut using a knife fashioned from glass or diamond (steel cutting edges are not used in TEM). These tiny thin sections can be treated with solutions of heavy metal salts, such as lead citrate or uranyl acetate, to enhance contrast of the section. These salts become deposited on different structures within the cell making them electron dense, and they appear darker in electron micrographs. Once treated, the sections are mounted on perforated copper grids and the grids inserted into an electron microscope. A beam of electrons passes through the section, creating an image that can be viewed on a fluorescent plate and used to create a negative or digital image.

Scanning electron microscopy is similar, in many aspects, to TEM methods, except that small pieces of tissue are specially coated with a thin layer of gold or palladium. Sometimes, the tissue is frozen and then fractured to reveal internal structure before the metal-coating step. The electron beam passes over the surface of the specimen, and the metallic coating reflects some of these electrons. The reflected electrons are detected and are used to create a three-dimensional image of the surface of the specimen. Such images can be recorded as negatives or digital images.