Most histological samples need preparation before microscopic observation; these methods depend on the specimen and method of observation.
Embedding Tissues are embedded in a harder medium both as a support and to allow the cutting of thin tissue slices.
Staining Biological tissue has little inherent contrast in either the light or electron microscope.
Staining is employed to give both contrast to the tissue as well as highlighting particular features of interest. When the stain is used to target a specific chemical component of the tissue (and not the general structure), the term
histochemistry is used.
Light microscopy staining on rat
trachea Hematoxylin and
eosin (
H&E stain) is one of the most commonly used stains in histology to show the general structure of the tissue. Hematoxylin stains cell
nuclei blue; eosin, an
acidic dye, stains the
cytoplasm and other tissues in different stains of pink. In contrast to H&E, which is used as a general stain, there are many techniques that more selectively stain cells, cellular components, and specific substances. A commonly performed histochemical technique that targets a specific chemical is the
Perls' Prussian blue reaction, used to demonstrate iron deposits in diseases like
hemochromatosis. The
Nissl method for Nissl substance and
Golgi's method (and related
silver stains) are useful in identifying
neurons are other examples of more specific stains.
Historadiography In
historadiography, a slide (sometimes stained histochemically) is X-rayed. More commonly,
autoradiography is used in visualizing the locations to which a radioactive substance has been transported within the body, such as cells in
S phase (undergoing
DNA replication) which incorporate tritiated
thymidine, or sites to which radiolabeled
nucleic acid probes bind in
in situ hybridization. For autoradiography on a microscopic level, the slide is typically dipped into liquid nuclear tract emulsion, which dries to form the exposure film. Individual silver grains in the film are visualized with
dark field microscopy.
Immunohistochemistry Recently,
antibodies have been used to specifically visualize proteins, carbohydrates, and lipids. This process is called
immunohistochemistry, or when the stain is a
fluorescent molecule,
immunofluorescence. This technique has greatly increased the ability to identify categories of cells under a microscope. Other advanced techniques, such as nonradioactive
in situ hybridization, can be combined with immunochemistry to identify specific DNA or RNA molecules with fluorescent probes or tags that can be used for immunofluorescence and enzyme-linked fluorescence amplification (especially
alkaline phosphatase and tyramide signal amplification).
Fluorescence microscopy and
confocal microscopy are used to detect fluorescent signals with good intracellular detail.
Electron microscopy For electron microscopy
heavy metals are typically used to stain tissue sections.
Uranyl acetate and lead citrate are commonly used to impart contrast to tissue in the electron microscope.
Specialized techniques Cryosectioning Similar to the
frozen section procedure employed in medicine,
cryosectioning is a method to rapidly freeze, cut, and mount sections of tissue for histology. The tissue is usually sectioned on a
cryostat or freezing microtome. The frozen sections are mounted on a glass slide and may be stained to enhance the contrast between different tissues. Unfixed frozen sections can be used for studies requiring enzyme localization in tissues and cells. Tissue fixation is required for certain procedures such as antibody-linked
immunofluorescence staining. Frozen sections are often prepared during surgical removal of
tumors to allow rapid identification of tumor margins, as in
Mohs surgery, or determination of tumor malignancy, when a tumor is discovered incidentally during surgery.
Ultramicrotomy under a
Transmission electron microscope Ultramicrotomy is a method of preparing extremely thin sections for
transmission electron microscope (TEM) analysis. Tissues are commonly embedded in
epoxy or other plastic resin. Very thin sections (less than 0.1 micrometer in thickness) are cut using diamond or glass knives on an
ultramicrotome.
Artifacts Artifacts are structures or features in tissue that interfere with normal histological examination. Artifacts interfere with histology by changing the tissues appearance and hiding structures. Tissue processing artifacts can include pigments formed by fixatives, shrinkage, washing out of cellular components, color changes in different tissues types and alterations of the structures in the tissue. An example is mercury pigment left behind after using
Zenker's fixative to fix a section. Formalin fixation can also leave a brown to black pigment under acidic conditions. ==History==