Dehydrating and embedding in paraffin - The world under the microscope

After fixation, the fixative must be washed out of the tissue. It was absolutely essential to avoid the use of tap water or aqua dest. now that the tissue is fixed, this is no longer important. Various literature have written that rinsing should take just as long as fixation. Most fixatives must be rinsed with tap water, but sometimes, as with Bouin, the tissue comes directly into the 70% ethanol.

As previously described, the tissue must be molded into paraffin to cut it properly. In order to get the paraffin into the tissue down to the cellular level, all the water must first be removed from the tissue. This dehydration takes place in a rising alcohol series. The series is necessary to gradually remove the water from the tissue, if it goes too fast it affects the tissue structure. Many types of alcohol series have been developed that all meet the requirements. The series used by the author is based on ethanol and isopropanol, which are easy to obtain. The series is: 50%, 70%, 85%, 95% and 100%. Especially the absolute ethanol is quite expensive to buy and is hygroscopic. The absolute isopropanol is cheaper and works fine. The tissue stays in each bath for 12 -24 hours depending on the size.

After all the water in the step 100% isopropanol has been removed from the tissue, the tissue is molded into paraffin. Before this is possible, an intermediate step (intermedium) must be made because isopropanol does not want to dissolve in paraffin. This step can be made with xylene or xylol. There are several substances that can act as intermedium such as: aniline, benzol, chloroform, isobutyl alcohol (butanol), tetraline, toluol, trichloroethylene, xylol and so on [2]. All these substances dissolve in both isopropanol and paraffin. The link page lists companies where the substances can be ordered. The tissue must remain in the xylene for 12 hours for small pieces and 24 hours for larger pieces. The pieces of tissue will look a bit glassy after this step (this is normal). After the intermedium step follows a step with a heated solution of 50% xylene and 50% paraffin. This step should take 24 hours for small pieces and 48 hours for larger pieces [2].

Paraffin is a mixture of saturated hydrocarbons with the general formula CnHn+2. For routine histology, paraffin with a melting point between 56-58°C is used. Modern "Paraffin for Histology" contains several plastic polymers which improve cuttability and have a high penetration capacity [2]. The author works a lot with "Paraplast plus". Perfectly sliceable with high penetration, however, it should not be heated above 62°C. All water has now almost disappeared from the tissue. The last residue of xylene is removed in 2 paraplast steps. Fill the cups and slowly heat them until the paraffin has just melted (make sure that paraplast does not exceed 62°C). In the first step, the tissue remains for 24 hours for small pieces and 48 hours for larger pieces. At the end of this step there will be a slight smell of xylene above the warm paraplast. The tissues will now be placed in the second cup of clean paraplast for another 24 or 48 hours. At the end of this step there should no longer be a smell of xylene. In case of doubt, another step with clean umbrellas may be taken. A longer stay of tissue in warm umbrellas has no harmful consequences. Poor cuttability on the microtome is often due to poor penetration with paraplast, which is often a consequence of incomplete drainage [2].

The tissues are now ready to be molded into blocks. An old-fashioned, but still widely used method is casting in a ring of aluminium after which the block is stuck onto a block of wood with a warm knife. On this site only stainless steel moulds will be used which will fit the cassettes exactly as described above. Because paraffin solidifies quickly, all materials must be ready for use. The tissue to be molded is best placed in a warm watch glass. The mold is placed on a steel plate. This is to allow the paraplast to cool down quickly. Fill the mold with a layer of paraplastic. Quickly position the tissue in the already coagulating paraplast. There is only a few seconds here to align. Warm tweezers can be beneficial here. The mold is filled with liquid paraplast up to the first edge. Place the cassette on the mold and fill it with paraplast. Soon the paraplast will start to solidify. Preferably place the hot mold in a cold environment (refrigerator). The faster the paraplast cools the smaller the paraffin crystals are, which improves the cutability.

Paraplast shrinks quite a lot and that's why there is a shrinkage drop in the cassette. This is fine. After about fifteen minutes everything is cured. Because the paraplast has shrunk it will fall out of the mold very easily. Now the block can be finished. Cut away the spouts and if necessary shrink the block. Not necessary, but practical. The paraplast can be reused again and all paraplast that is in a cut has to be dissolved later in xylene. There must be enough paraplast to keep the tissue in place to keep it strong enough. Number the blocks and archive them. They can be kept for years.

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[1] Prof. Dr. Peter Böck (1989, 17., neubearbeitete auflage), Romeis Mikroskopische Technik, München. Verleger Urban & Schwarzenberg. Chapter 5, 'Auswaschen der Präparate' blz 114.