Advanced Histological Techniques

Although secondary fixation in osmium tetroxide provides some areas of electron density, this is usually not sufficient to provide high contrast, high definition images. A number of staining techniques are available to enhance the contrast of areas of interest. These fall into two major categories. Positive stains deposit electron dense material on the area of…

The ultrathin sections required in TEM are cut with knives of glass, diamond or sapphire. These materials produce extremely hard, ultrasharp edges, but they are brittle and subject to damage. Glass knives are produced as needed by fracturing. Sapphire knives and diamond knives may be purchased. The high cost of diamond knives possibly makes resharpening…

Sections for TEM must be less than 80 nm thick in order to allow at least 50% of the electron beam to penetrate the sample. This can only be accomplished by using resins for embedding (epoxy, acrylic or polyester) which requires a modification of the processing protocol. Graded alcohol baths (typically 20, 40, 70, 90…

The most popular fixatives for TEM work are aldehydes and osmium tetroxide. Aldehyde based fixatives react with amines and other nucleophiles in the tissue, most notably lysine and arginine, generating cross-linked proteins. The cross linking action of these fixatives stabilizes the cytosol, preserving cellular structures. Aldehydes do not react with most lipids, so membrane components…

The resolution of a microscope is limited by the wavelength of light passing through the sample. For visible microscopes using 400 nm light (blue light), the limit of resolution is one half the wavelength, or 200nm. This is some two to three orders of magnitude larger than many cellular structures. Electrons, like photons, have wavelike…

Light microscopy makes use of primarily two detection systems for immunohistochemistry – fluorescence and enzyme labeling, while electron microscopy relies on the deposition of electron dense materials at the site of antibody binding. Techniques for light microscopy are discussed below. EM is covered briefly in the next section. Immunoflourescence The conjugation of a fluorescent dye…

The antibody systems used in Immunohistochemistry can be broadly divided into two types, direct and indirect. With the direct method the visualizing agent is attached directly to the antibody that will bind with the antigen. The direct method is technically and theoretically straightforward, and yields results sufficient for many studies. Its sensitivity is limited by…

Immunohistochemistry is the application of antibody/antigen interactions to provide information about biological systems. The body’s response to the introduction of a foreign agent, known as the immune response, results in the production of antibodies which bind the offending material. Antibodies bind tightly and specifically to an “epitope” (one specific structure) on an “antigen” (foreign molecule…