General Overview of Histology
Histological techniques provide a visual means for the examination and analysis of cell/tissue physiology and morphology at the microscopic level. Histology represents a broad technology, invaluable for studying and understanding the microscopic three-dimensional organization, structure, and function of cells and tissues and is especially useful for the diagnosis and understanding of disease at the cellular level. The process first involves isolation and fixation of the cells/tissue of interest. Fixation preserves the structure and morphology of the specimen throughout the harsh conditions of dehydration, clearing, embedding, sectioning and staining.
Native, unfixed tissues will undergo the natural processes of putrefication, decomposition, and autolysis. Autolysis is rapid in tissues that are rich in enzymes, such as the liver, brain and kidney, and is less severe in fibrous tissues such as collagen. Fixation functions by preventing sample degradation while at the same time stabilizing and preserving the ultrastructure and morphology characteristic of the native tissue. Fixation must convert the soluble contents of the cell (i.e. proteins, carbohydrates, nucleic acids, and lipids) into insoluble networks that are resistant to the harsh subsequent processing steps. This procedure can either be accomplished through chemical or physical means.
Physical methods for fixation include heat and dessication. Both methods are harsh and are not suited for the preservation of fine ultrastructure. Heat works by denaturing proteins, much like cooking an egg. Heat fixation is commonly used for preparing smears of blood cells or bacterial cells for microscopic examination.
Chemical fixation often involves the use of reactive aldehydes, most commonly formaldehyde and glutaraldehyde, for the preservation of microstructure. Fixation should occur as rapidly as possible, thereby preventing the natural procersses of decay. Formaldehyde, being the smallest of the aldehydes, can rapidly penetrate into samples, providing a quick, mild fixation. Glutaraldehyde, a larger molecule with two functional aldehyde groups penetrates more slowly, but typically provides a more rigid crosslinking of the proteins and a more stable fixation. Often the two aldehydes can be used in conjunction. Since glutaraldehyde generally gives a more rigid fixation and preserves fine microstructure better than any other fixative, it is the preferred fixative for specimens in electron microscopy.
The most commonly used metallic ion in fixation is osmium tetroxide. It is typically employed as a secondary fixative in electron microscopy. Osmium tetroxide is known to form cross-links with proteins and there is some agreement that osmium tetroxide reacts with unsaturated lipids, preserving membrane structures. Osmium tetroxide is used for preservation of fine structures in electron microscopy and is effective for small (2-3 mm3) specimens.
Non-aqueous chemicals like acetone and alcohols are also commonly used as fixatives. Alteration of the structure of proteins brought about by methanol and ethanol is primarily due to disruption of the hydrophobic interactions that contribute to the maintenance of the tertiary structure of proteins. Hydrogen bonds appear to be more stable in methanol and ethanol than in water so that while affecting the tertiary structure of proteins, these alcohols may preserve their secondary structure and any associated epitopes. Alcohol fixation can cause distortion of nuclear detail and shrinkage of cytoplasm. If fixation is prolonged, the alcohols can remove histones from the nuclei and later extract RNA and DNA.
Other fixatives include acetic acid, picric acid, heavy metal ions salts like mercuric chloride, potassium dichromate, and zinc sulfate. Many fixatives are used in conjunction and the choice of fixative is highly dependent on the type of tissue or cell type to be examined and the type of stain to be used. Good resources for fixation protocols can be found at:
Ultimately specimens will need to be cut into thin sections for examination on a microscope. This requires that the specimens be embedded in some type of support medium that can be properly mounted and sectioned with a microtome. Proper embedding usually requires that the sample is first dehydrated, as many of the embedding mediums are non-aqueous and cannot infiltrate tissues that contain water. Dehydration is best performed by sequential immersion in solutions of increasing ethanol concentrations (i.e. 25% ethanol, 50% ethanol, 75% ethanol, 90%, 95%, 100%). Often the dehydrated samples are then “cleared” of the alcohols by incubating them in organic solvents like xylene, toluene, or benzene. These chemicals function to remove the alcohols and make the sample receptive to complete infiltration by the embedding medium, as they are miscible in both alcohols and many embedding resins. Once specimens have been properly dehydrated and cleared, they are ready for embedding in media like paraffin wax or epoxy resin. Paraffin is commonly used for routine histological analysis with light microscopes. Electron microscopy requires that sections be cut extremely thin (i.e. 60-100 nm) and demands the use of a sturdy media like epoxy resin. For specific protocols on sample processing/embedding see the following links:
An alternative to dehydration and infiltration of the specimen with epoxy or paraffin is cryosectioning. In this manner, the specimen can be isolated from the animal, immersed in a cryoprotectant/mounting solution like OCT compound, and flash frozen in liquid nitrogen. These frozen samples can then be cut into sections on a cryostat (microtome in a refridgerator) and the sections can be mounted on glass slides and then fixed/stained in any manner. Cryosectioning is extremely fast and can be useful for techniques that are sensitive to the harsh conditions of dehydration and infiltration like immunostaining and enzyme-activity staining.
The cellular material within tissues is typically transparent and without large differences in optical refractive indices, the cells and subcellular structures that compose a tissue are not readily apparent. To aid in the visualization of cells, extracellular matrices and subcellular components, a wide variety of stains and staining procedures has been developed. Staining relies on the binding, deposition, or partitioning of a colored substance with a particular type of substance within the specimen by chemical or physical means. By far the most common stain for routine histology is the hematoxylin/eosin (H&E) staining technique. Hematoxylin, when combined with certain metal ion mordants forms a dye-metal complex that acts as a basic dye, staining the negatively charged phosphate groups of nuclear DNA. The blueish-brownish color of the cellular nuclei after hematoxylin stain of the specimen is often countertstained with a dye called eosin. Eosin is an acid dye that effectively binds positively charged arginine, histidine and lysine side chains of cellular proteins. In this manner eosin can stain the cytoplasm of cells a pinkish-reddish color, it also stains erythrocytes and collagen. Go to these links for general protocols on H&E staining:
Below is a picture of an H&E stained specimen. Note the different cell types and morphology present in the section
H&E staining is a general stain to visualize cell morphology in tissue sections. There are countless other stains that can be extremely specific in terms of what is stained, i.e. connective tissues, cartilage, carbohydrates, lipids, certain microorganisms, etc. For a comprehensive list of stains go to:
Enzyme histochemistry is a technique for directly visualizing the presence of specific enzyme activities within a tissue. The presence of certain enzyme activities can correlate with pathology and enzyme histochemistry is often used in diagnosis of diseases affecting skeletal muscle. Enzymes present within tissues are very sensitive to processing conditions and usually these specimens are prepared by cryosectioning and mild fixation in cold acetone, alcohols, or formaldehyde solutions. Some common techniques include staining for ATPase or acid phosphatase activities.
Another alternative to the classical histological stains is immunohistochemical staining. Immunostaining involves the use of antibodies for the specific recognition and staining of a protein within the cells of a sample. Like enzyme histochemistry, the staining is sensitive to harsh conditions, as the specific epitopes of the protein targets must remain intact. Mild fixatives like cold alcohols or formaldehyde usually preserve these epitopes for recognition by antibodies. The antibodies used to bind the protein target are either directly labeled with an enzyme or fluorochrome for direct microscopic visualization, or they are themselves bound by a labeled secondary antibody. Below is an example of immunostaining of an expressed protein with a fluorescent (FITC) labeled antibody against the protein in tissue culture cells. In (A) the protein is expressed in the cytoplasm of the cells. In (B) the protein has been engineered such that it is fused with a nuclear localization signal, and the corresponding fluorescence of the immunostain is primarily nuclear. These cells were fixed in 3.7% formaldehyde prior to immunostaining.
Elecron microscopic analysis of specimens requires ultrathin sectioning in epoxy resins. Samples are typically fixed in both glutaraldehyde and osmium tetroxide prior to dehydration and embedding in the resin. These sections are then mounted on TEM grids and typically stained with heavy metal salts like uranyl acetate, phosphotungstic acid, or lead citrate to provide contrast. Different parts of the cells take up the stains differently and the electron-dense metal atoms are opaque to the electron beam, creating a dark contrast on the micrograph. Below is an example of adenovirus infected cells visualized under the TEM. The picture was taken from Gaden et al. 2004 JVI 78(13); 7227-7247. Note the direct visualization of adenoviral particles on the cell membrane.
Histology is a very complicated science and it embodies an extremely large set of methods and principles for the preparation and visualization of biological samples. Presented above is a brief overview of some of the principles. For specific protocols on prepping and staining a tissue of interest one should refer to Histotechnology: A Self-Instructional Text by Frieda L. Carson or Theory and Practice of Histotechnology by Dezna C. Sheehan and Barbara B. Hrapchak. These reference represent comprehensive and useful guides to histology. A good webpage for specific sample fixation, processing, and staining with protocols is: