By : Columbia University
In order to study tissues with a microscope they must be preserved (fixed) and cut into sections thin enough to be translucent. The process of fixation is briefly described in the next section. Fundamentally it consists of a chemical or physical method of killing the tissue and yet retaining characteristic peculiarities of shape and structure. Following fixation, blocks of tissue must be cut into thin sections. One way is to make a firm block by freezing fresh or fixed tissue. Other techniques involve dehydration in alcohols and infiltration with paraffin, or some similar agent - a process called embedding. Sections 3 to 10 microns (3 to 10 thousandths of a millimeter) in thickness are cut on steel knives mounted in an instrument called a microtome, which has a precise mechanical advance.
For electron microscopy the sections are considerably less than one ten-thousandth of a millimeter (0.1 micron, µm) thick. This is accomplished by embedding the tissue in a plastic such as Epon or araldite (epoxy resins) and cutting on special ultramicrotomes equipped with a fine mechanical or thermal advance. Sections are cut with glass or diamond knives and mounted on copper mesh grids.
In work with the light microscope, it is difficult to recognize the various components of cells and tissues without differential staining. The stains may react chemically or physically and a wide variation is possible. The staining method can be altered to suit the needs of the examiner in order to accentuate certain tissues or organelles.
Finally, in order to preserve the section which has been made from a block of fixed tissues and stained, it is mounted on a glass slide and covered with a thin cover glass by means of a transparent substance which hardens and seals the preparation to make it permanent. Some tissues are stained and then mounted. More often the tissue is placed on the slide first and then stained. The mounting medium used to attach the coverslip must have a refractive index similar to that of the glass slide and cover slip to prevent distortion.
Slide Preparation Techniques
Histology Techniques - Fixation
This process has two phases:
1) the coagulation or precipitation of the various components of the tissues and cells and
2) their preservation in a state as nearly as possible like the living condition by forming stable chemical compounds.
The first phase carries with it an intrinsic source of difficulty and error. The precipitation may be uneven and cause deposits to form where no structure existed in the living cell. These are called "fixation artifacts". The second phase also carries a source of difficulty because the compounds formed by some fixatives will not take up some stains.
It has not been possible to find an ideal fixative that
1) penetrates quickly,
2) renders all parts of all cells permanent and
3) allows the use of all kinds of stains.
The reason for this is not difficult to understand. The cell is a highly complex mixture of proteins, carbohydrates and fats. The ideal fixative would not only have to form stable compounds with all of these, but also render them insoluble both in fat solvents and in water. Some fixatives not only fail to preserve certain parts of the cell but actually dissolve or destroy them. For example, acetic acid destroys mitochondria. Moreover, some fixatives change the shape and relationship of parts of a tissue by shrinkage.
This is a good general fixative. Its effect is to cross-link membrane proteins by forming covalent bonds. It is made by dilution of commercial formaldehyde (which is a 40% solution of formaldehyde gas in water) in an aqueous phosphate buffer. The usual strength is 10% (or 4% of the gas). It penetrates rapidly, causes little distortion, does not destroy any of the cellular constituents and can be followed by almost all staining methods. It hardens the tissues very slowly, however, and does not protect them from the shrinking agents employed in embedding and sectioning. For this reason it is often combined with other fixing agents.
Osmium tetroxide (OsO4) preserves the cell in a form closer to the living than any other fixative. Its great disadvantage is that it penetrates poorly and cannot be followed by many stains. It is also used as a stain because it blackens fat and various lipid-containing materials such as the myelin sheaths of nerve fibers, and makes them insoluble both in water and in fat solvents. Osmium tetroxide solution, in various buffers, is a standard fixative for electron microscopy.
Histology Techniques – Embedding
Since water and paraffin do not mix, the first step in embedding with paraffin is to replace the water in the tissues with a solvent that is miscible with paraffin. Following are the steps in paraffin embedding:
- Dehydration - It is the first part of the process. It is usually accomplished by transferring the block of tissue through a series of alcohol-water solutions beginning with 50 percent and running up to water-free or absolute alcohol.
- Clearing - The alcohol is replaced by Histoclear (a non-toxic substitute for xylol) or cedar oil, which is readily soluble in alcohol, and in turn, is replaced by melted paraffin.
- Embedding - The actual embedding takes place when the paraffin- infiltrated tissue is placed in fresh paraffin and the latter allowed to cool. It is important to remember that the xylol and other solvents will dissolve the fats of the tissues unless they are fixed by some special chemical such as osmic acid.
Celloidin is dissolved in equal parts of absolute alcohol and ether. The tissue is dehydrated in alcohol in the same way as for paraffin except that it is transferred from absolute alcohol to a dilute solution of celloidin. As the alcohol and ether evaporate, they are replaced by more concentrated celloidin. It is finally hardened in chloroform and stored in 80 percent alcohol. It is a much longer process than paraffin but causes much less shrinkage and distortion. It is used especially in examination of the eye and brain.
Introduction of epoxy embedding media has greatly reduced artifacts due to shrinkage and also has allowed thinner sectioning than was possible with paraffin. The thinner sections (approximately 1 u) may be viewed after staining with the light microscope or may be sectioned thinner and examined by electron microscopy.
Histology Techniques - Staining
Stains react in two general ways: 1) They combined directly with the tissue, or 2) they require that the tissues be treated first with an anchoring substance or mordant. Very few stains can be relied upon to color with the desired selectivity or intensity unless carefully controlled. This may be accomplished by stopping at the desired intensity or removing excess with another reagent.
Selective stains have been found for many of the different parts of the cell and for characteristic elements in the tissues. Much of the selective action is due to the fixation and previous treatment as well as to the subsequent staining and differentiation.
Impregnation is not really a staining process but it is considered as one of the staining methods. The tissues are first placed in a solution of the salt of a heavy metal. The metal is precipitated as a black deposit about certain structures. These stains are especially used for study of neurons and glia of the central nervous system.
Basic and Acid Dyes
Basic dyes are cationic. They form salts with tissue anions (components that carry a net negative charge), especially the phosphate groups of nucleic acids and the sulfate groups of the glycosaminoglycans. Basophilic is the term used to designate the components of a cell or tissue, which take up the basic stain rather than the acid stain of a combination. Nuclei are basophilic.
Acid dyes are anionic. They form salts with cationic groups in cells and tissues, particularly the ionized amino groups of proteins. Acidophilic or oxyphilic is applied to parts, which show a greater affinity for acid dyes. The cytoplasm is usually acidophilic. Eosinophilic components are cationic compounds that have an affinity for that acid dye.
Following are some terms related to dyes:
- Mordants - A mordanting substance is considered part of the stain, and in this way it may change the reaction of the stain. For example, hematoxylin is an acid, but as it is almost always used in conjunction with alum or iron (the mordant) it becomes a basic stain.
- Amphophilic - It is a term used to indicate that the tissue stains with both the basic and the acidic dyes.
- Neutrophilic - No special affinity for either the basic or acidic components of a dye.
- Metachromasia - It refers to the production of a color during staining which is different from the original color of the staining solution. Mast cell granules will stain a reddish-purple with toluidine blue. Metachromasia is pH dependent. Many substances are only metachromatic when stained as frozen sections. Usually they must be viewed immediately, if not sooner.