Water Soluble Wax
Water soluble wax, Carbowax, polyethylene glycol and PEG are all terms used to describe the same material. Chemically they are polyethylene glycols of varying degrees of polymerisation and molecular weights, or polyethylene glycol fatty acid esters, such as polyethylene glycol monostearate. At molecular weights above 1000 they are solids which closely resemble paraffin wax in texture, and are suitable as embedding media, causing less shrinkage than does paraffin wax. As the molecular weight increases the materials become harder with higher melting points. Like paraffin waxes, polyethylene glycol is usually a mixture of materials with varying molecular weights, due in this case to varying degrees of polymerisation, rather than a single molecular entity. When purchasing, the molecular weight has to be specified to ensure the correct material. Molecular weights of 1500 or so are the most commonly used. Both polyethylene glycol and its stearic acid ester are considered safe, being used industrially and as food additives.
The infiltration is very simple. Since polyethylene glycols are water soluble, dehydration and clearing are not needed. After fixation, the tissue is first immersed in 50% polyethylene glycol in water, then in multiple changes of polyethylene glycol alone (100%), at temperatures just above the melting point of the polyethylene glycol chosen, and for long enough to infiltrate the tissue, usually an hour or so in each. Or they may be first infiltrated with polyethylene glycol then by the stearate ester. The blocks are cast in the last of these. Once the tissue has been placed into 100% polyethylene glycol, water is avoided as it may soften the block. This means that the blocks must be allowed to cool and harden without being immersed in iced water. Instead they are left to cool at room temperature or placed into a refrigerator. It should be emphasised that during the infiltration process all the water is removed and replaced with polyethylene glycol. The final blocks are cast after having been infiltrated with pure polyethylene glycol and no water remains in the tissue.
While the infiltration is relatively simple, flattening and picking up cut sections presents some difficulties. With paraffin, the commonest procedure is to float sections on warm water to soften and flatten, then to pick them up by immersing the slide into the water beside them. This cannot be done with polyethylene glycol sections as the polyethylene glycol dissolves away when it contacts the warm water, and currents may cause the tissue to be disrupted. If the tissue is cohesive enough to withstand the dissolution of the supporting polyethylene glycol, the addition of a small amount of a surfactant to the flotation bath may help, but it means there will be no material left within and around the tissue for protection during the baking on process. Some success may be achieved with small amounts of various fluids being placed on individual slides and sections floated on that, then immediately drained in an attempt to minimize removal of the support medium. Otherwise it is necessary to place the section directly onto the slide by one edge and pull it flat. With practice, and a degree of manual dexterity, flattened sections can be obtained. Sections may also be collected in water (with the addition of a surfactant to minimise currents) then treated as free floating sections similar to frozen sections.
Since this is an aqueous processing medium and avoids dehydrants and clearing agents, lipids are not removed. This means that very fatty tissues will retain their fat content and, depending on the amount of fat present, may be difficult to section. Other tissues will retain the finer lipid droplets within and around cells, and these can be demonstrated. Polyethylene glycol is not lipophobic and is miscible with molten triglycerides, although this is of little practical significance.
Keep in mind that this is a water soluble medium, so "dewaxing" sections can be done with water, or any other fluid in which polyethylene glycol dissolves, i.e. almost anything, including staining solutions. If an aqueous mounting medium is used for coverslipping, it becomes a completely aqueous technique from fixation to the final stained slide. Of course, that is not obligatory.
Due to their susceptibility to water, polyethylene glycol blocks must be stored free from humidity. This may be difficult in practice, so it is more convenient to coat the blocks with a thin film of paraffin wax for storage. the wax is broken off and removed when the blocks are to be sectioned.
Due to the difficulty of flattening sections onto slides and the problems associated with storage in a moisture free environment, polyethylene glycol processing has not become popular and remains a specialised procedure, not suitable for routine diagnostic use.
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