Tissue Preparation for Paraffin Embedded Tissue

Formalin-fixed tissue is processed with a standard procedure, which includes dehydration, clearing, infiltration, and embedding. Dehydration is necessary in the preparation of tissue blocks for embedding in a nonaqueous medium, such as paraffin, celloidin, and some plastics. These media will not infiltrate tissue that contains water. There are two ways in which the dehydrating agents act to remove water. Some reagents are hydrophilic and attract water from tissue, whereas other reagents dehydrate by repeated dilution of the aqueous tissue fluids. Most dehydrating agents are alcohols (e.g., ethyl alcohol, methyl alcohol, isopropyl alcohol, and butyl alcohol). Acetone is a very rapid-acting and less-expensive dehydrator.

Clearing agents also are referred to as dealcoholization agents; their primary purpose is to remove alcohol used for dehydration and to make the tissue receptive to the infiltration medium. Xylene is the most widely used clearing agent. Xylene is a flammable reagent and is considered a hazardous substance, so the waste product must be either recycled or disposed of in an appropriate manner.

After dehydration and clearing, tissue must be infiltrated with a supporting medium, which also is called embedding medium. Paraffin wax is the most popular medium because the tissue blocks may be processed in comparatively short period of time, serial sections are easily taken and routine and most special stains are easily done. Water-soluble waxes, celloidin, plastics, agar and gelatin and 30% sucrose are examples of other embedding media (19,20).

A wide variety of processors are available. The two major systems used by processors are the open system and the closed system. The open system does not use vacuum, whereas the closed system uses heat and vacuum. The closed system is computerized and can be programmed to change the solutions at desired intervals. The following is an example of solutions and times for routine overnight processing with a closed system that is used in our laboratory utilizing the Thermo Shandon Hypercenter XP processor (see Note 3):

1. A total of 3 h in alcoholic formalin.

2. 40 min each in six changes of dehydrant.

3. 40 min each in three changes of clearing agent.

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