Histology Articles

Histological staining involves the use of dyes to highlight specific intra- or extracellular elements within tissue. A vast array of dyes and associated staining protocols exist in use. Each dye is targeted toward different cellular structures. The response to a given protocol can vary among samples. Many protocols are up to 100 years old and…

Artifacts that appear in stained slides may result from a number of causes including improper fixation, the type of fixative, poor dehydration, improper reagents, or poor microtome sectioning. The presence of a fine black precipitate on the slides, often with no relationship to the tissue (i.e., the precipitate appears adjacent to tissues or within interstices…

Once embedded, tissues are cut into thin sections ready to be placed on a slide. This is done with a microtome, an apparatus for feeding the blocks past an ultrasharp blade with micron level precision. Paraffin blocks can be sectioned with high-carbon steel blades. Plastic blocks (methacrylate, araldite, or epon) are sectioned with glass or…

For mechanical support during the sectioning process, tissue must be infiltrated with an embedding medium. The usual embedding media are paraffin for light microscopy and an epoxy resin for EM samples. Paraffins are available that differ in melting point and hardness. Some products contain added plasticizers to make the blocks easier to cut. In general,…

The step following dehydration is called “clearing” and consists of replacing the dehydrant with a substance that will be miscible with the embedding medium (paraffin). The term “clearing” comes from the fact that the clearing agents often have the same refractive index as proteins. As a result, when the tissue is completely infiltrated with the…

Dehydration is usually carried out by transferring the tissue through solutions of increasing alcohol concentration, until 100% alcohol is reached. Sometimes the first step is a mixture of formalin and alcohol. Other dehydrants can be used, but have major disadvantages. Acetone—though fast—is a fire hazard, so it is safe only for small, hand-processed sets of…

Once fixed, the tissue must be treated to allow the cutting of the thin sections required for viewing under the microscope. The procedures designed to prepare the tissue for sectioning are collectively known as tissue processing. First, the sample is dehydrated by immersion in a series of aqueous alcohol solutions gradually moving to pure alcohol.…

Fixatives are among the most hazardous substances used in life science research. Work with these substances under the hood wearing gloves, lab coat and safety goggles. Formaldehyde is a suspect cancer hazard and a strong sensitizer. It is harmful if inhaled or absorbed through the skin. High exposures may be fatal. Formaldehyde can cause blindness…

Fixation protocols are usually straightforward. The tissue is cut to dimensions suited to the rate of penetration of the particular fixative and placed in the fixative solution. The number of factors affecting the fixation process includes buffering, penetration, volume, temperature and concentration. In fixation pH is critical. This is especially the case with formaldehyde, where…