Intranuclear birefringent inclusions, apparently paraffin, are often observed in paraffin sections which are dehydrated in acetone instead of alcohol after staining. These inclusions are not removed by prolonged immersion of the sections in xylene or other fat solvents at room temperature. They may be removed from unmounted sections by clearing these sections in xylene after the usual hydration and alcohol dehydration, either before or after staining. They may be removed from resin-mounted sections, but not completely so, by warming the sections in an oven at 58° C. for 15 minutes.

When paraffin sections of routine pathological material, which were stained with haematoxylin-azure-eosin and dehydrated in acetone, are examined under polarized light, unstained birefringent inclusions of varying size and shape are seen within occasional nuclei. A much larger number of inclusions can be shown by mounting duplicate sections unstained in resin immediately after deparaffinization in xylene or by mounting the sections in glycerine jelly after staining (fig. 1). These inclusions are not removed by prolonged (18-hour) immersion of the sections at room temperature in xylene, dioxane, petroleum ether, or gasoline.

Such intranuclear birefringent inclusions can be found in the majority of paraffin sections of a wide variety of tissues and organs, both human and animal, fixed in various fixatives. These inclusions are generally found in small nuclei occurring in the midplane of the section. They are particularly numerous in lymphocytes of the spleen and of the appendix, in stromal cells of the endometrium, and in endothelial cells of blood vessels. They do not appear in sections of tissue embedded in carbowax (Firminger, 1950) or ester wax (Steedman, 1947) or in frozen sections of fresh or fixed tissues, mounted either in aqueous or resinous media.

The following studies were undertaken to determine the nature of these inclusions. Paraffin sections mounted in glycerine jelly and containing numerous birefringent inclusions were placed in an electrically heated microscope stage (Kofler micro hot stage, A. H. Thomas Co.) and heated gradually while being continuously observed under polarized light. The majority of the inclusions disappeared at temperatures between 54° C. and 58° C., while a few persisted up to 63° C. When the sections were now cooled, the inclusions began to reappear at 61° C., and the majority reappeared at temperatures between 55° C. and 50° C. At this last temperature the inclusions seemed to be present in their original numbers. In resin-mounted sections the inclusions disappeared at a slightly lower temperature, 51° C. to 55° C., with a few remaining to 57°.C., and upon cooling only a small percentage of the inclusions reappeared.

In order to compare these results with the behaviour of paraffin, which is known to be birefringent in its solid state, duplicate unmounted sections were placed in the same apparatus and the paraffin in the periphery of the sections observed with increasing temperatures. The birefringence of the paraffin was found to disappear at temperatures between 53° C. and 57° C., with rare crystals persisting up to 60° C. This temperature at which the birefringence was lost is the melting-point of the paraffin used.

To test further the relationship between the melting-point of paraffin and the disappearance of the intranuclear birefringent bodies, the above procedure was repeated on sections of tissue embedded in a low (45° C.) meltingpoint paraffin. In this case, the temperature of disappearance of the inclusions was 44° C. to 47° C., with rare inclusions persisting to 48° C. The meltingpoint range of the peripheral paraffin, determined microscopically as above, was 43° C. to 45° C., with rare crystals persisting to 46° C.

Since molten paraffin will dissolve fat-soluble colouring agents, attempts were made to colour the inclusions with oil red O by means of the supersaturated isopropanol method (Lillie, 1948) at several temperatures. At room temperature and at temperatures up to about 50° C., the inclusions remained unstained. When the sections were stained for 15 minutes at 65° C. or for more prolonged periods at slightly lower (55-60° C.) temperatures, red globules of varying size were found in the majority of nuclei containing birefringent bodies (fig. 2).

To study the effect of the usual xylene-alcohol-water-alcohol xylene sequence of staining procedures upon these inclusions, serial sections of a tissue-block containing numerous inclusions were passed through this sequence. At each stage in the sequence a slide was removed, a cover-glass applied, and the slide examined immediately under polarized light. The birefringent bodies were found to disappear completely as the sections were cleared in the last xylene, following the hydration and dehydration of the sections. When sections were dehydrated in acetone instead of alcohol, the subsequent clearing in xylene removed most but not all of the inclusions.

Several other methods of removing these inclusions were investigated. When unmounted paraffin sections were immersed in hot (70° C.) xylene for 30 minutes, most but not invariably all of the inclusions were removed. When resin-mounted sections, either stained or unstained, were dipped for a few seconds in hot (70° C.) water or, alternatively, heated in a 58° C. oven for 15 minutes, most but not all of the inclusions were removed.

Since the above findings show that these inclusions are birefringent at room temperature, lose their, birefringence at approximately the same temperature as the melting-point of the paraffin used for embedding, are stained by a fatsoluble colouring agent at temperatures above the melting-point of paraffin, and are absent in frozen, carbowax, or ester gum sections, these bodies are believed to be paraffin. Apparently the fixed nuclear membrane of some cells prevents the complete removal of paraffin by xylene or other, fat solvents applied at room temperature. It is not known why xylene, even at room temperature, will remove all intranuclear paraffin after the hydration and alcohol dehydration of sections. The partial persistence of intranuclear paraffin following the routine haematoxylin-azure-eosin stain of this laboratory is apparently due to the brief acetone dehydration of sections required by this technique, in which alcohol cannot be used for dehydration following staining.

This study suggests that paraffin sections intended for examination under polarized light should preferably be dehydrated in alcohol rather than acetone after staining. If acetone dehydration is unavoidable, the majority of the persisting intranuclear paraffin inclusions can be removed by placing the resinmounted sections in an oven at 58° C. for 15 minutes. Paraffin sections intended for very critical study of small nuclei should preferably be passed through the usual xylene-alcohol-water-alcohol-xylene sequence first, and then hydrated again before staining, in order to remove all of the intranuclear paraffin before the stain is applied.

Firminger
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H. L.
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1950
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25
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Lillie
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R. D.
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1948
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Histopathologic Technic, p. 159. Philadelphia (Blakiston Co
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Steedman
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H. F.
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1947
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Quart. J. micr. Sci
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88
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