Histology Fundamentals

Here are a few guidelines which you can use to process fixed tissue using Histo-Clear and Histo-Clear II: Histo-Clear Automated Tissue Processing Schedule Process Bath 18 Hour Cycle (Time in Hours) 24 Hour Cycle (Time in Hours) 70% ethanol 1.5 2 80% ethanol 1.5 2 95% ethanol 1.5 2 Absolute alcohol 1.5 2 Absolute alcohol…

Most dyes used to visualize the membranes and organelles of the cell are water soluble. The embedded wax must therefore be removed prior to staining. This is done by effectively reversing the tissue processing schedule. There are literally thousands of staining protocols and procedures in use. As an example, one of the most common stains,…

To preserve and support a stained section for light microscopy, it is mounted on a clear glass slide, and covered with a thin glass coverslip. The slide and coverslip must be free of optical distortions, to avoid viewing artifacts. A mounting medium is used to adhere the coverslip to the slide. Aqueous based mounting media…

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.…