Diagnostic Electron Microscopy on Reembedded ("Popped Off") Areas of Large Spurr Epoxy Sections

P. Anthony di Sant'Agnese and Karen L. De Mesy-Jensen, University of Rochester Medical Center, Department of Pathology Box 626, Rochester, NY 14642

Reprinted by permission of Karen L. de Mesy-Jensen
First appeared in Ultrastructural Pathology, 6:247-253, 1984

Introduction

The removal of tissue or cells from glass slides by inverted BEEM capsule "pop-off" techniques for the purpose of ultrastructural studies has been used in exfoliative cytology (1-5) and with paraffin-embedded material (6). The pop-off technique was first used to remove cell culture monolayers from petri dishes for electron microscopic studies (7).

Recently, a pop-off technique has been directly applied to plastic sections (8). We have developed a pop-off technique in which small areas of thick Spurr epoxy sections cut from large blocks are removed directly from glass slides and subsequently thin sectioned. The tissue is processed rapidly considering the large size of the section. This procedure has greatly increased the efficiency of our laboartory and improved our results.

Materials and Methods

Human surgical specimens were cut into pieces measuring approximately 1.0 x 1.0 x 0.1 cm, and some were as large as 1.5 x 1.5 x 0.2 cm. The majority of the tissues were fixed in a 4% paraformaldehyde/1% glutaraldehyde solution, pH 7.3, at 4ºC for 12-24 h.

To make 1000 ml:

40 g paraformaldehyde in 980 ml distilled water (60ºC).
Add 3.2 g NaOH and cool to room temperature.
Add 11.6 g NaH₂PO₄ and dissolve completely.
Add 20 ml 50% glutaraldehyde.

The tissues were then processed with continuous agitation as follows:

1. Wash in Sorensen's phosphate buffer (two changes, 15 min each).
2. Postfix in 1% OsO₄ for 3 h.
3. Wash in Sorensen's phosphate buffer (two changes, 15 min each).
4. Dehydrate 15 min each in increasing concentrations of acetone 50%, 80%, 95%, and then 100% (three changes, 15 min each).
5. Infiltrate with 1:1 100% acetone-Spurr (9) for 30 min using a DER ranging from 5-10 g. (Some specimens were infiltrated and embedded in Spurr with 1% Dow Corning 200 fluid silicone of 0.65 centistoke vsicosity as per Langenberg. (10)).
6. Infiltrate 3 h or overnight in 100% Spurr.
7. Embed in Spurr using plastic JB-4 molding trays of 12 x 6 x 5 mm
   (#H1514) or 12 x 16 x 5 mm (#H1513) sizes and aluminum block holders
   (#H1512) and polymerize overnight at 70ºC.

Nonperfused animal research material was minced to approximately 1 mm cubed pieces while bathed in 2% glutaraldehyde-Sorensen's buffered fixative. The tissues were then fixed in this solution for 24 h at 4ºC and processed in a similar manner to the surgical specimens, except the solution times were cut in half and infiltration was overnight. Twenty to thirty cubes of tissue were embedded in 12 x 6 x 5 mm JB-4 molding trays with block holders and polymerized overnight at 70ºC.

Sections were cut dry at 1-2.5 um thickness on a Sorvall-type JB-4 "Porter-Blum" microtome (#H1500) using Ralph-Bennett knives (11) at a clearance angle of 1-2 degrees. The knives were made on a LKB 2078 histoknife maker. A waxed metal T-adapter (LKB) for Ralph-Bennett knives was used to hold the knife in place. The sections were transferred to distilled water on glass slides, heated on a Corning hot plate at 70ºC to induce spreading, and then dried slowly to the slides at 50ºC. Before staining or "popping off," the sections were further heated at 100ºC for 1 h on a hot plate.

Sections were stained with a sequential basic fuchsin-methylene blue stain developed in our laboratory, (12) which differentially stains a wide variety of substances. The slides were then studied to select areas of interest for thin sectioning. An adjacent unstained section was retained for dotting with a water-insoluble felt tip pen directly over the area of interest. Because of the OsO₄ postfixation the area of interest could usually not be readily identified by light microscopy (if not, a stained section can be used directly with no obvious adverse effect on the ultrastructure). A size 3 BEEM capsule filled with partially polymerized (viscous) Spurr (DER 5 g) epoxy resin was inverted over the area marked. The Spurr must be partially polymerized or else the dot will dissolve. The capsule on the slide was polymerized for 3 h at 90ºC. The slide was then heated on a Corning Hot Plate-Stirrer at high heat for a few seconds until the plastic just began to vaporize. The slide was then removed from the hot plate and the capsule was gently rocked back and forth to pop off the section. The procedure was repeated if the capsule did not come off with gentle rocking.

The block face was then trimmed to a mesa around the dot. Care must be taken to align the block face properly: the initial sections contain the specimen, which is only 1-2.5 um in thickness. Up to 10-20 or more silver thin sections can usually be obtained from a 2-um piece of tissue. The sections were stained with alcoholic uranyl acetate-lead citrate and examined using a Hitachi HS-8 electron microscope.

Discussion

Sampling problems are a major drawback to efficient and productive transmission electron microscopic studies. A wide variety of techniques have been developed to try to overcome these problems. Most of the techniques have in common the embedding of tissue in large plastic blocks. The similar techniques using "mesa trimming" (13-14), destroy all the remaining tissue in the block except for the area to be thin sectioned. Techniques that attempt to circumvent this problem are generally complex and tedious. They range from core-drilling of the block (15), the difficult re-embedding of very thick sections (16,17), to the cutting of extra large thin sections, which are divided during sectioning (18). A pop-off technique has been recently described for small single plastic sections (8). The pop-off technique in this paper is an extremely simple and efficient method of localizing areas for ultrastructural study. Large plastic-embedded sections of diagnostic human surgical specimens as well as minced diagnostic or research material embedded as many small cubes per large block (20 or more) may be used.

We have found the processing procedure for large tissue blocks of human surgical specimens described in the "Methods and Materials" to give good ultrastructural preservation. The relatively rapid technique (given the large size of the specimens) allows for one-day processing with overnight polymerization. A modified McDowell's fixative (19) is used as recommended by Hayat (20) for large tissue sections. With the use of this "pop-off" technique, diagnostic surgical material can be studied most effectively. Large sections are highly advantageous in that the tissue can be studied with the maintenance of tissue relationships. Common sampling problems with surgical material such as necrosis, autolysis, and poor fixation can be avoided as much as possible. Furthermore, areas or individual cells of the lighest diagnostic value can be preselected by light microscopy. For example, the finding of widely scattered individual cells with suspected viral inclusions by light microscopy can be confirmed directly by electron microscopy. Individual cells or groups of cells with probable rhabdomyoblastic differentiation can be selectively studied ultrastructurally to confirm (by the finding of thick and thin filaments and/or Z-band material) a possible diagnosis of rhabdomyosarcoma. In some poorly differentiated rhabdomyosarcomas, cells containing these dignostic features may be extremely difficult to locate by conventional electron microscopic techniques. Perfused research material can also be embedded in large blocks, and difficult to find structures or cells can be identified and studied ultrastructurally. The large size of the tissues probably does not allow for absolutely optimal preservation for a variety of reasons, including extraction of materials during the longer processing times and differential time exposures to the vrious solutions at different levels of the tissue. However, the pop-off technique can still be used to advantage with optimally prepared diagnostic or animal research tissue minced into small 1 mm cubed pieces. Twenty or more pieces can be embedded in one large block, sectioned, stained, and studied under the light microscope in order to select the piece that is most representative. The piece can then be popped off, thin sectioned, and studied under the electron microscope. A research control rat liver is shown to demonstrate the lack of distortion and excellent ultrastructural preservation of optimally fixed and processed minced tissue that has been popped off.

The hardness of the large plastic block can be varied considerably by varying the amount of DER 736 from 5-10 g in the final plastic mixture. The softer the block the longer the glass knife will last and the easier it is to cut, but there must be a reasonable compatibility of hardness between tissue and block for optimal sections. We generally use a DER of 7 g but occasionally use lower amounts for bony or calcified specimens and higher amounts for very soft tumors. By always popping off with a DER of 5 g, good stability under the electron beam is acheived using uncoated grids. A plastic mixture with DER of 7 g or more is not very stable and reinfiltration during pop-off must stabilize the section. We have also found that the addition of 1% silicone (Dow Corning 200 fluid silicone of 0.65 centistokes) to the plastic mixture (10) increases glass knife life and allows for thinner sections to be cut dry due to decreased sticking properties. The addition of silicone did not affect the pop-off or the ultrastructural preservation of the tissue.

The only minor problem associated with this pop-off technique is the added skill and patience necessary to cut the thin sections. The skill lies mainly in the very accurate alignment of the tissue block face to the knife. When this is done properly, up to 10-20 silver sections can be obtained from a single 2-um embedded section.

References

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