Clearing Tissue Sections
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 clearing agent, it becomes translucent. This change in appearance is often used as an indication of the effectiveness or completeness of the clearing process.
The most common clearing agent is xylene. Xylene is reasonably cost effective and works well for short-term clearing of small tissue blocks. Long-term immersion of tissue in xylene results in tissue distortions. Toluene is better at preserving tissue structure and is more tolerant of small amounts of water left behind in the tissues than xylene. However, toluene is more expensive than xylene and more toxic, so toluene is less commonly used. Chloroform has been used in some applications, but it is a severe health hazard, acts slowly and may lead to sectioning difficulties. Methyl salicylate is safe and effective, though rarely used due to costliness.
Orange oil based clearing agents—such as National Diagnostics' Histo-Clear—offer the best clearing action with the lowest hazard rating of all xylene alternatives. Histo-Clear is excellent for preserving fine tissue structure, and can often be used in place of xylene with no alteration of protocol. In using a product containing orange oils, it is important to use a product such as Histo-Clear which has been rigorously purified then stabilized. Orange oils that are neither pure nor stable can break down to produce compounds which will interfere with staining procedures.
Most tissue processing is done using automated machines that carry out the steps automatically. Tissues coming off a tissue processor are in a plastic box ready for the embedding stage.
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