Techniques In Neuroscience: Paraffin Wax

Paraffin wax is one of the most popular reagents for infiltration and embedding as it is hard enough to give adequate support to the tissues but soft enough to allow fine sectioning using a microtome blade. There are various degrees of melting points between 56 and 60 degrees but it is not soluble in water, therefore the tissue must be already processed before embedding.

Preparatory steps

Following from the point above, firstly, the tissue must be adequately dehydrated. We succeed in this step by gradually immersing the tissue in higher and higher concentrations of alcohol, such as 70% followed by 90% and ultimately 100% pure alcohol. Each laboratory will have its own standard procedures which may vary.

Secondly, the alcohol must be removed as well as it does not interact well with either water or paraffin. This is a process called “clearing” which is linked to the clearing agents (xylene, Histo-Clear), which are substances miscible with alcohol and paraffin wax.

• Xylene is cost effective and ideal for small paraffin wax blocks for short amounts of time as long term immersion would cause distortions in the tissue being studied.
• Toluene is used less often due to higher levels of toxicity and the higher price point.
• Chloroform is sometimes used but, again, there are issues with high levels of exclusivity.
• Methyl salicylate is not as toxic but is one of the more expensive agents, making it seldom used.
• Histo-Clear is one of the best agents to use as it has a high level of purity and low health risk associated.

The third step is called infiltration and it refers to replacing the clearing agent with a cell-supporting medium, most commonly molten wax, in preparation for the embedding process. Paraffin wax is miscible with some clearing agents but not with alcohol, which should have been removed completely in step two. Finally, at this stage the tissue has been appropriately prepared and embedded in the medium. Cooling is necessary at this stage in order to obtain a block which is solid enough for ribboning. The cooling process is usually done at 4 degrees Celsius by using cooling plates or ice dishes.

Cooling the tissue increases the level of quality for the prepared tissue and will lead to better cutting quality and should also reduce processing time. Variations in the temperature used and method of cooling will also affect the cutting quality and most laboratories will have preferred methods.

In manual embedding procedures, cracking may occur if the cooling process is done too quickly or if the paraffin block was still hot when it was subjected to cooling. Therefore, it would be ideal to cool to room temperature first and then cool further to 4 degrees.

Factors which affect overall quality of microtome sectioning A microtome is a tool used to cut very fine (~0.6mm diameter) slices of medium in sections or “ribbons” which are appropriate to be studied under a microscope. At step three a few factors such as melting point, hardness, stickiness and brittleness are considered in choosing the type of paraffin wax. Paraffin wax is soluble at room temperature but will melt at temperatures between 65 degrees and 70 degrees. The higher the melting point, the harder the paraffin, which will make sectioning more difficult. A lower melting point will usually denote a softer paraffin which is easier to section. Another factor is that sufficient wax must be poured in the cassette in order to ensure that the wax levels will not recede during the cooling process, leading to gaps between the block and cassette.

Storage of prepared paraffin sections

Exposure to air will lead to oxidation and will affect the final quality of the staining of the tissue and the quality of the results of studying proteins.

Mouse brain protocol

The protocol for brain tissue should be as described above, as for all other visceral organs, however, the brain tissue is softer and care should be taken not to solidify during the infiltration process.