Histology involves the use of a set of techniques to examine the morphology, architecture and composition of tissues. The desired tissue sample is first dissected and fixed. Then the tissue is embedded in a compound in preparation for either plain sectioning or cryosectioning. After the tissue has been sliced, sections are mounted on a slide in preparation for component identification via staining, immunohistochemistry, or in situ hybridization.
Fixation is performed to preserve the structure of the tissue. This process provides rigidity to the tissue, making it
easier to section. There are a variety of forms of fixation, including "fresh frozen," where the tissue is not actually
placed in fixative. Common fixatives used include formaldehyde (formalin) and glutaraldehyde. Ethanol, acetone, and
paraformaldehyde are often used for cryopreserved specimens. While samples are often fixed before cryopreservation, this
is not always necessary.
The choice of fixative depends on the staining technique (histological staining, immunohistochemistry, in situ hybridization)
to be used later. Most histological staining methods, but not all, allow the use of formalin. Immunohistochemistry can
be performed on paraffin/plastic embedded or cryopreserved specimens that have been fixed, but some antibodies will not be able
to bind to the target molecules after the formalin fixation and paraffin embedding processes.
Once placed in the fixative, covalent bonds are formed within the amine groups of the tissue, crosslinking it. A tissue sample
is fixed for only a short period of time, usually overnight, as longer fixation times may cause artifacts to be apparent at
high levels of magnification. Once samples have been fixed, they can be kept indefinitely at room temperature. Once fixation
has been completed, the sample is embedded prior to sectioning.
Before sectioning, tissue samples are embedded in a material with similar mechanical properties. This step allows
the tissue to be cut easily.
If a tissue is to be sectioned with a microtome, it can be embedded in paraffin, or in plastic resin if the tissue is
particularly hard. After fixation, tissues that are to be paraffin-embedded are dehydrated by first using graded
ethanol solutions, then graded xylene solutions, then finally liquid paraffin. The graded solutions gradually expose the
sample to changes in hydrophobicity, minimizing damage to cells. After a short time in the liquid paraffin, the tissue is
placed into a mold with more paraffin. The wax is allowed to solidify, forming a block that can be held in a
microtome.
Cryosectioning is often used if either immunohistochemistry or in situ hybridization is to be performed, or if there is a concern that heat from paraffin embedding will damage the tissue. Samples that are to be cryosectioned are placed in a polymer solution, often polyethylene glycol (PEG) in water. This solution desiccates the tissue in a similar manner as a hypertonic solution. The tissue and solution are placed in a mold, which is then frozen to form a block that can be cryosectioned.
Samples embedded in paraffin are first mounted in a microtome. The microtome holds a sharp blade and is controlled by a crank that is turned to bring the paraffin block closer to the blade. As the crank is turned further, the blade cuts slices of paraffin, which containing tissue. The microtome can be adjusted for width and angle of cut. After sectioning, the slices can be placed on a slide.
Samples embedded in a polymer solution are sectioned using a cryostat, which allows thin sections to be cut at a low temperature.
After several slices of the paraffin-embedded tissue have been sectioned, the slices are removed from the blade and floated atop a warm water bath to smooth out the sample. The slices are teased apart and floated onto a slide (Poly-L-lysine (PLL) modified slides will retain tissue sections better than plain slides). After the slides have dried, they are placed in an oven to "bake" the paraffin. They can then be stored until processing.
After cryosectioning, the slices are also mounted on a slide. The mounting medium will then be allowed to melt, which smoothes out the section on the slide.
Different staining techniques are available to preferentially color components of interest in tissues. Some
frequently used stains are shown below.
One especially common stain is Hematoxylin and Eosin (H&E), which colors the nuclei dark blue (hematoxylin) and the
remaining cell components pink (eosin).
Mason's Trichrome is a three-color stain that stains cell nuclei red, collagen green or blue, and cell cytoplasm red.
Verhoeff and Van Gieson are two stains for elastin, often performed over H&E. These two stains color only mature elastin black. Verhoeff is shown below.
The Von Kossa Stain signals calcification in tissue (in black).
Safranin-O stains glycosaminoglycans red.
Immunohistochemistry uses antibodies to identify tissue components of interest. It can be used to identify both cell
types and extracellular matrix components. Both enzyme-based and fluorescently based immunostaining techniques exist.
Enzyme-labeled samples are viewed in a bright field after a series of steps. Fluorescent staining tends to be more sensitive
than enzyme-based staining, which should be considered when selecting the staining technique.
The first steps in immunohistochemistry are to section and mount the tissue. This staining technique is dependent on the
fixative used, because the antibody epitope may be masked during fixation. Cryosectioning is usually a better option, but
many labs use paraffin embedded sections. After mounting, the sample is incubated with a
blocking agent, such as bovine serum albumen (BSA), in order to prevent non-specific binding. After blocking, the
sample is incubated with a primary antibody, selected to bind to the antigen of interest on the tissue, and rinsed.
The sample is then incubated with a secondary antibody, which binds to the primary antibody, acting as an
amplifier of the visual signal. If fluorescence staining is used, the secondary antibody is generally labeled with
a fluorescent tag, and the antibody binding can therefore be visualized after this step. For enzyme-based immunostaining,
the secondary antibody is tagged with biotin, and additional steps must be carried out prior to visualization. It is possible
to label the primary antibody; however, there is generally not enough primary antibody bound to the sample to be visible.
Steptavidin linked to horseradish peroxidase is applied to the tissue section. Diaminobenzidine (DAB) is then added,
resulting in a brown-colored precipitate forming where the antibody has bound. Enzyme labeled sections are often
further counterstained with hematoxylin to enhance visualization prior to viewing under a light microscope.
Once example of immunohistochemistry is Proliferating Cell Nuclear Antigen (PCNA) staining, which labels the nuclei of
proliferating cells. Fluorescence is more sensitive when viewing this stain, but it lacks visual context for the labeled
cell components, as can be seen below.
In situ hybridization analyzes the gene expression of the histological specimens. In this technique, a fluorescently or radioactively labeled probe is applied to a tissue section in which the cells have been permeabilized. The probe is preselected to bind a DNA or RNA sequence of interest, to aid in identifying the genetic makeup or physiology of the cell. After wash steps to remove unbound probe, bound probe can be visualized either under fluorescence or by using an appropriate radioactivity detector.
The cartoon below shows the steps involved in Fluorescence In Situ Hybridization (FISH).
