DIETHYLENE GLYCOL DISTEARATE EMBEDDING AND ULTRAMICROTOME SECTIONING FOR LIGHT

MICROSCOPY1

HERNANDO SALAZAR,~ Department of Anatomy, Washington University School of Medicine, St . Louis 10, Missouri

ABSTRACT. Diethylene.glyco1 distearate can be used as an embedding medium for light microscopy. Two infiltration changes of about 6 hr each in the melted wax (melting point 47- 52 C) are required before the final embedding which is done in 00 gelatin capsules for section- ing in the ultramicrotome by the procedure used in electron microscopy. Serial sections 1-2 p thick can be cut without difficulty. No cooling devices are necessary for trimming and sectioning at laboratory temperature. Sections rarely become detached from the slides. The staining char- acteristics of the tissues are the same as when embedded in paraffin. For fluorescence microscopy, essentially the same procedure is followed. Tissues are not distorted and the intracellular struc- tures are well preserved.

Diethylene glycol distearate was used first in histological work by Orton and Post (1932) and later by Cutler (1935), with satisfactory results when tissues were dehydrated prior to infiltration. Inasmuch as this compound diffuses in water, Orton and Post thought that it might be used for embedding tissues directly from the fixative and thus eliminate distortion caused by dehydrating agents. However, the tissues that they embedded by this procedure suffered shrinkage after a few days.

Steedman (1947) published the results on numerous methods of embedding, particularly with ester waxes, and concluded that a mixture of 73 gm of diethyl- ene glycol distearate, 10 gm of diethylene glycol monosterate, 4 gm of ethyl cellulose, 5 gm of stearic acid and 8 gm of castor oil had given the best results.

Later, in 1948, Smyth and Hopkins used successfully the mixture proposed by Steedman for an improved demonstration of glycogen. Chesterman and Leach (1 956) modified Steedman’s embedding medium by purifying the glycol stearates.

In 1957, Steedman tested more than 50 different compounds and mixtures, and recommended polyethylene glycol 400 distearate as the best. This polyester wax has been used by Sidman et al. (1961) as the routine embedding medium in their laboratory.

Lacy and Davies (1959) used diethylene glycol distearate in a technic for the demonstration of insulin by fluorescent antibodies, with excellent results.

The purpose of the present report is to give details for using diethylene glycol distearate as a generally applicable embedding medium for conventional light and fluorescence microscopy, with special reference to sectioning in the ultra- microtome.

This investigation was supported in part by United States Public Health Service Grant RG- 3784.

Rockefeller Foundation Research Fellow, on leave of absence from National University of Colombia School of Medicine, Bogotb, Colombia. Present address: Universidad Del Valle, Facultad de Medicina, Cali, Colombia.

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14 STAIN TECHNOLOGY

MATERIALS AND METHODS

The technics of embedding and sectioning that will be described were used particularly on hypophyses of mice and rabbits. In general, the tissues were re- moved from the animal 1-2 rnin after death and fixed in modified Heidenhain’s SUSA, made by adding 1 volume of saturated aqueous picric acid to 9 volumes of the original SUSA mixture. The specimens weie dehydrated by direct transfer to 95% ethanol, 2 changes of 6 hr each or overnight; followed by 2 changes of abso- lute ethanol of 3 hr each; then cleared in xylene, 2 changes of 2 hr each.

Em bedding. Diethylene glycol distearate (Kessler Chemical Co., Inc., Phila- delphia 35, Pa.) was kept melted in a 57-60 C oven for 24 hr before using, in or- der to reduce its viscosity and facilitate its penetration into the tissues. However, it should not be kept in the oven for longer periods of time or heated at higher temperatures because it will decompose. Although the wax has a melting point of 47-52 C it is stable at 60 C for 24-72 hr, and no evidence of damage of the tissues is seen when they are embedded in an oven temperature of 57-60 C. Tis- sues were infiltrated by a first change of the melted wax for 6 hr and a second one of 6-16 hr. At the final change, the tissues are transferred to 00 gelatin cap- sules (the same type used for electron microscopy) that have been filled with melted wax, placed in suitable wells drilled in wooden blocks preheated for 15 min, and replaced in the oven for 1-2 hr. The capsules are then transferred to another wooden block, previously cooled for 15 rnin in a refrigerator, so that solidification starts at the bottom of the capsule; thus the formation or trapping of air bubbles near the specimen is avoided. For the latter step a metalic device which would provide for a faster solidification of the block could also be used.

Tissues dehydrated by freeze-drying (Lacy and Davies 1959) are placed directly in gelatin capsules containing the melted wax. Each piece is dropped on the surface of the wax and, when all the capsules are ready, they are placed again in the oven for 10-15 rnin to melt the wax that has started to solidify at room temperature. They are next transferred to a desiccator and moderate vacuum is established for approximately 5 min (vacuum ovens can be used for this pur- pose). When the vacuum is broken the pieces of tissue sink to the bottom of the capsules. The wooden block with the capsules is returned to the oven for 15-30 min to ensure complete infiltration. Finally, the capsules are transferred to a cooled wooden block and solidification allowed to occur at room temperature or in a refrigerator.

Sectioning. A Porter-Blum ultramicrotome model TM-1 and glass knives were used. The entire gelatin capsule can be easily removed from the wax block with a razor blade, but it is advisable to remove only the tip, so that the block is pro- tected from shattering when placed in the holder. The block may be either trimmed to obtain a desired size or area, or sectioned without trimming. Sec- tioning itself is done by the same procedure and technic used in electron mi- croscopy. Sections are cut with ease at l, 2, or 3 p. Thinner or thicker sections can also be cut without difficulty, if necessary. Sections are floated on distilled water in a knife trough and, by means of a small piece of bond paper, transferred to regular glass slides and again floated on distilled water. Without draining

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GLYCOL DISTEARATE EMBEDDING 15

the water, the slide is placed on a 37-45 C hot plate, where the section will flatten and adhere to the slide. Egg albumen can be used for microscopy by or- dinary light but it is fluorescent and, therefore, unsuited for fluorescence light- ing.

Staining. Dewaxing, hydrating and staining procedures are the same as those used for paraffin-embedded tissues: In this study, the following stains were made: periodic acid-Schiff-orange G; aldehyde fuchsin-hematoxylin; hematoxylin-eosin; alcian blue-periodic acid-Schiff-orange G; Heidenhain’s “azan” reaction; and periodic acid-Schiff-hematoxylin (figures 1 and 2).

When the fluorescent antibody technic is used, sections are dewaxed by im- mersion in xylene for 2-3 min and air dried without treatment with alcohols. T h e staining with the conjugated fluorescein-globulin is made as usual.

DISCUSSION T h e results obtained confirm the reports made by previous investigators who

used diethylene glycol distearate embedding (Orton and Post 1932, Cutler 1935, Lacy and Davies 1959). T h e preservation of the cellular structures is very good and there is minimum distortion or shrinkage caused by the embedding process. On the other hand, manipulation of the blocks for sectioning, trimming, etc., is facilitated by the consistency and the melting point of the wax. Although paraffin used in histological preparations usually has a melting point higher than that of diethylene glycol distearate, i t frequently requires the use of ice or a cold en- vironment to prevent softening of the block and to ensure consistently good sec- tioning, especially when sections less than 5 p thick are to be cut. Polyethylene glycol 400 distearate, as reported by Sidman e t al. (1961), must be cut in a cold atmosphere to obtain ribbons of thin sections. For this reason, they have sug- gested the use of dry ice on a support above the microtome and thus produce a current of cold air to cool the block and the knife. With diethylene glycol di- stearate it is not necessary to cool the block or the knife. Sections as thin as 1 p can be cut easily at a laboratory temperature of 25-28 C, without any cooling device-a great advantage of this material. Detachment of the sections from the slides is an extremely rare occurrence.

Inasmuch as the trough made on a glass knife for floating sections is rela- tively small, ribbons of a large number of sections cannot be accommodated. However, excellent results can be obtained when individual sections are cut as a series. As many as 80 successive sections have been cut without loss. Slow sec- tioning and change of the glass knives every 10-20 sections is recommended to avoid compression, disruption and scratching of the sections.

If desired, larger blocks can be made in the usual manner for sectioning on a Minot rotary microtome with either regular steel knives or wide glass knives held in the knife holder by means of a simple adaptor (A. D. Chiquoine, personal communication).

T h e brittleness of diethylene glycol distearate does not cause inconvenience when the described procedure is used. If for any reason the block is broken in the specimen area, it can be easily re-embedded in another capsule.

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16 STAIN TECHNOLOGY

FIG. 1. Mouse anterior hypophysis. Elftman fixative. Diethylene glycol distearate embedding.

FIG. 2. Rabbit anterior hypophysis. SusA-picric acid fixative. Diethylene glycol distearate em-

FIG. 3. Rabbit anterior hypophysis. SusA-picric acid fixative. Paraffin embedding. PAS-orange

FIG. 4. Rabbit anterior hypophysis. SusA-picric acid fixative. Paraffin embedding. PAS-orange

AIcian blue-PAS-orange G stain. 3 p section. x 400.

bedding. Aldehyde fuchsin-hematoxylin stain. 1.5 p section. x 1000.

G-hematoxylin stain. 4 p section. X 400.

G-hematoxylin stain. 4 p section. X 1000.

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GLYCOL DISTEARATE EMBEDDING 17

Occasionally some batches of diethylene glycol distearate become contami- nated with dirt after use. It is then necessary to filter the melted wax through coarse paper before using it.

I t is concluded that the following features of this compound make advisable its broader use in histology: (a) preservation of the tissue architecture and cellu- lar integrity of specimens embedded in this wax, (b) its easy handling and sec- tioning qualities, ancl (c) preservation of the conventional staining characteris- tics.

ACKNOWLEDGMENT

I wish to express my thanks to Dr. Jack Davies for his suggestions and as- sistance during the course of this investigation.

REFERENCES

CHES~EKMAN. If’., and LEACH, E. H. 1956. A modified ester wax for embedding tissues. Quart.

CUTLER, 0. I. 1935. Embedding in glycol stearate. Arch. Path., 20, 445-6. LACY, P. E., and DAVIES, J. Demonstration of insulin in mammalian pancreas by the

fluorcsrent antibody method. Stain Techn., 34, 85-9. ORTON, S. T., and POST, J. 1932. Some experiments with a new embedding material. Bull.

Neurol. Inst., 2, 302-11. SIDMAN, R. L., MOTTLA, P. A., and FEDER, N. Improved polyester wax embedding for

histology. Stain Techn., 36, 279-84. SMYTH, S. D., and HOPKINS, C. A. 1948. Ester wax as a medium for embedding tissue for the

histological demonstration of glycogen. STEEDMAN, H. F. Quart. J. Micr. Sci., 88, 123-33. - 1957. Polyester wax. A new ribboning embedding medium for histology. Nature, 179,

J. Micr. Sci., 97, 593-7.

1959.

1961.

Quart. J. Micr. Sci., 89, 431-6. 1947. Ester wax: A new embedding medium.

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