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…

Mercury Based Fixatives Mercurials contain mercuric chloride. Their method of tissue fixation is poorly understood. While not penetrating tissue well and causing some tissue hardness, mercurials are fast and provide excellent nuclear detail. They are commonly used to fix hematopoietic and reticuloendothelial tissues. Alcohol Fixatives Alcohols, including methyl alcohol (methanol) and ethyl alcohol (ethanol), are…