Unembedded, aldehyde-fixed tissue, sectioned for transmission electron microscopy

J O U R N A L O F U L T R A S T R U C T U R E RESEARCH

79, 250-272 (1982)

Unembedded, Aldehyde-Fixed Tissue, Sectioned for Transmission Electron Microscopy D A N I E L C . PEASE

Department of Anatomy, School of Medicine, University of California, Los Angeles, California 90024 Received July 16, 1981, and in revised form January 21, 1982 It has been demonstrated that it is feasible within limits to section a wide variety of unembedded, glutaraldehyde-fixed tissues thinly enough for transmission electron microscopy. Much cytological detail is preserved. The success of the method is thought to depend mainly upon glutaraldehyde adequately cross-linking cytosol proteins so that cellular components are not only held in place, but the tissue is sectionable after it is air dried. A polyvinyl-acetate emulsion in the form of carpenter's white glue is used as an external support for tissue blocks. If necessary, this can be rendered insoluble by the addition of a small quantity of serum albumin, subsequently cross-linked with glutaraldehyde. Glass knives are advisable for sectioning, and sections are floated on a layer of water reaching the knife edge with a minimal meniscus. Since alkaline lead stains have proven to be quite destructive to unembedded tissue, and other positive stains have not been found that are particularly effective, partially neutralized phosphotungstic acid, used as a negative stain, is the principal staining technique employed. Because it is thought that the technique ultimately may prove to be most useful for immuuocytochemical applications, emphasis has been placed upon studies of tissues fixed only with glutaraldehyde. If tissue is secondarily fixed with osmium tetroxide and/or treated with uranyl salts, however, tissue blocks then have superior sectioning properties, and can be more effectively and positively stained.

In recent years, the vastly improved technology for obtaining highly specific antibodies has stimulated great interest in the potentialities of immunocytochemistry. There remain problems, however, with the intracellular penetration of antibody molecules, particularly when these are coupled with the very large macromolecules used as markers for electron microscopy. The most elegant approach to the penetration problem has been the development of ultracryomicrotomy. Although originally conceived by Fernfindez-Morfin as early as 1950 and 1952, it was Bernhard and Nancy (1964) who first succeeded reasonably well in sectioning glutaraldehyde-fixed tissue at -30°C, while supported with gelatin. Later refinements by Bernhard and Leduc (1967) demonstrated its potential quality and stimulated subsequent appropriate commercial instrumentation. Its practicality was notably advanced, particularly by the subsequent work of Bernhard and Viron (1971),

Christensen (1971), and Tokuyasu (1973, 1978, 1980). It must be admitted, however, that the procedure to this day is fraught with sufficient difficulty so that it has not so far been widely employed. Instead, it would seem that most investigators have found it easier to partly destroy cell membranes in tissue blocks by detergent action, or by alternating freezing and thawing cycles, so as to expose the cellular contents to the external milieu. Generally such preparations finally have been processed conventionally with attendant extraction by organic solvent action. It is generally recognized, and indeed sometimes deliberately planned, that many of the cytosol proteins are lost and/or destroyed by such procedures. Another approach is to consider the possibility of ultrathin sectioning of unembedded tissue. It is the purpose of the present report to demonstrate the feasibility, within limits, of achieving just that. Although an

25O 0022-5320/82/060250-23502.00/0 Copyright O 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.

SECTIONING U N E M B E D D E D TISSUES

aldehyde fixation is employed, only an extracellular support is required. Organic solvents are avoided. In spite of air drying, much fine structure is well preserved. It is anticipated that antigenic binding sites not destroyed by the initial aldehyde fixation will be exposed after the sectioning is completed. Furthermore, the full complement of cytosol proteins should be preserved and available for study. The present work has been predicated upon the demonstration of Farrant and McLean (1969) that cross-linking a concentrated serum albumin gel with glutaraldehyde, with subsequent drying, could produce an adequate external support for sectioning small biological objects at room temperature. They examined erythrocytes, small algae, bacteria, and isolated mitochondria. Later, Nicolson (1971) used the same procedure to obtain interesting sections of isolated chloroplasts. Their experiments with albumin gel suggested that the proteins of the cytosol of many cell types might also be adequately cross-linked with glutaraldehyde so as to permit ultrathin sectioning. The recent report of Griffiths and Jockusch (1980), who studied immunocytochemically both liver and muscle tissue using the Farrant and McLean technique, expressed this conclusion, and they compared their results with cryosectioned material. In fact, two other groups of investigators also have applied this procedure in immunocytochemical tissue studies. First, Kraehenbuhl and Jamieson (1976) explored the technique with an epithelial tissue (pancreas), and used the sections obtained for successful reactions with ferritin-labeled antibodies. Subsequently, Kraehenbuhl and co-workers extended these investigations (1977), and then joined Papermaster and other colleagues in applying the methodology to studies of retinal receptors (1978a,b). It is interesting that there has been a surprisingly long, but tenuous, history of other efforts to obtain ultrathin sections of unembedded tissue without employing low

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temperatures. As long ago as 1946, Fullam and Gessler had the idea that they might be able to section unembedded, frozen-dried tissue with the ultrahigh-speed microtome that they had developed. Admittedly, this was an unsuccessful effort, terminating with pulverized samples. Later, Gil~v (1956, 1958)had considerable success in sectioning osmium tetroxide-fixed striated muscle which was supported by dried gelatin. At about the same time, independently, Fernfindez-Morfin and Finean (1957) fairly satisfactorily sectioned nerve myelin that had been fixed either with osmium tetroxide or potassium permanganate, and which also was supported by hardened gelatin. Additionally, in the early years of ultrathin sectioning, an effort by Richards et al. (1942) was made to employ high molecular weight polyethylene glycols. If fixation techniques had been adequate at the time so that cytomembranes might have been preserved, such large molecules presumably would not have penetrated cellular compartments, and thus might then simply have provided an external support. Penetration may have occurred in at least some instances, however, so one cannot necessarily speak of such tissue as being truly unembedded. Particularly interesting in relation to the recent and present work is a report by Borysko, published obscurely in 1963. This described efforts that he had made to section unembedded tissue, generally following formaldehyde or osmium-tetroxide fixation. Air-dried tissue was simply cemented to supports with a commercial adhesive. In this w a y he succeeded remarkably well in sectioning the ordered collagenous fibrils of tendon, and also he was able to present a few reasonably good micrographs of cellular tissues. There followed a partially successful attempt by Koller and Bernhard (1964) to repeat and extend Borysko's work. Difficulties led them, however, to first dry aldehyde-fixed tissue with either liquid COz or NzO at their critical points. Since N20 is partially miscible with water,

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it h a d t h e a d v a n t a g e o f a v o i d i n g c o n v e n tional dehydration. Such critical point dried tissue then was sectioned quite well without embedding. They even explored the possibility of collecting dry cut sections w i t h t h e a i d o f a n e l e c t r o s t a t i c field. I t rem a i n e d f o r T r a n z i e r (1965), h o w e v e r , w o r k i n g in B e r n h a r d ' s l a b o r a t o r y , t o p r o v i d e a n excellent demonstration of sectioned, unembedded tissue that had not been exposed to any organic solvents. His prepar a t i o n s i n v o l v e d air d r y i n g t h i c k c r y o s e c tions on suitable supports, followed by fixation, usually with aldehydes, and finally ultrathin sectioning. The present study represents a broadly b a s e d e f f o r t to e x p l o r e s o m e o f t h e t e c h n i c a l p r o b l e m s i n v o l v e d in s e c t i o n i n g a n d otherwise handling unembedded tissue. T h e p r a c t i c a l i t y o f t h e g e n e r a l m e t h o d , as w e l l as its l i m i t a t i o n s a r e c o n s i d e r e d . P o r tions of this work have appeared previously in a b s t r a c t f o r m ( P e a s e , 1980a,b). A l s o , a n immunocytochemical application has been p r e s e n t e d ( P e a s e et al., 1981), a n d h a s b e e n s u b m i t t e d f o r p u b l i c a t i o n ( P e a s e e t al., 1982). MATERIALS AND METHODS

Fixation Tissues that have been particularly studied to date include rat lung, kidney, nerve roots, pancreas, and liver, as well as frog retina. Some effort also has been devoted to more complexly organized organ tissues including rat intestinal wall, trachea, and uterus. Since the primary concern was not so much morphologically optimal fixation as subsequent preparative steps, these tissues have most commonly been fixed by the simple immersion of thin (-1 mm) tissue slabs in aldehyde solutions, although in the case of the lung, trachea, gut, and uterus, infusion and perfusion techniques have been employed. In general, the primary fixative has been 2% glutaraldehyde added to Hanks solution, with the pH adjusted to be slightly on the alkaline side of neutrality. The primary fixation was usually either for 3 hr or overnight. Since immunocytochemical applications have been an ultimate objective of these investigations, we have been interested in preserving tissue in as nearly a native state as possible. Thus, fixation has generally been limited to aldehydes, and secondary fixation omitted.

Some effort has been made to explore minimally permissible fixation. A 30-min exposure to 2% paraformaldehyde in Hanks solution has proven to be adequate for the sectioning of kidney and pancreatic tissue, as has been a 30-min exposure to 1% paraformaldehyde, plus 0.2% glutaraldehyde, in the case of these tissues, as well as for frog retina. After the primary fixation was deemed to be complete, small tissue blocks always were passed through 4 to 6 successive -5-min changes of neutralized physiological saline solution to remove excess aldehyde before processing them further. The use of glutaraldehyde as the principal primary fixative of course did not preclude at times secondary fixation with such other agents as osmium tetroxide (2%, 1 hr), tannic acid (2%, 1-2 hr), and uranyl salts (2% uranyl acetate, I hr, or 0.2% uranyl magnesium acetate, overnight).

Tissue Support Early experiments followed the technique of supporting specimens as proposed by Farrant and McLean (1969) in which small tissue blocks were soaked in 30% serum albumin. This was subsequently concentrated so that it gelled. After partial drying, a secondary exposure to glutaraldehyde then cross-linked the gel to provide an external support for the specimens. After washing out the glutaraldehyde and fully drying the blocks, lung, pancreas and kidney tissue could indeed be sectioned, and it was evident that much fine structure had been preserved. One problem with the Farrant and McLean technique was soon apparent; the albumin filled the stroreal compartments of the tissue, and ultimately stained essentially as did the nonfibrous contents normal to connective tissue or, for that matter, the contents of the cytosol. It was obvious that for tissue studies a contrasting external support system would be preferable. The presumption was that this should be a colloid that could not possibly penetrate plasma membranes. Polyvinyl-acetate emulsion is such a substance, readily available in the form of "white carpenter's glue." We tested several easily available brands of white glue (including "Elmer's Glue-All," Borden) without noting significant differences. We settled on a product manufactured by "Wilhold Glues" of Santa Fe Springs, California. This glue is said to contain 2% plasticizers and 1% unspecified hydrocarbons besides the polyvinyl acetate and water (Gosselin et al., 1976). Our own measurements indicated that it consisted of 56% solids. Its original pH was 4.4, but ammonium hydroxide could be, and sometimes was, added to increase the pH at will, although we had the impression that the water resistance of dried samples deteriorated somewhat if the pH was elevated much above 6. Its particle size, again our own determination, was not at all uniform, the smallest particles being of the order

SECTIONING UNEMBEDDED TISSUES of 0 . 1 / x m in diameter (Fig. 3), and the largest ones in e x c e s s of 5 / x m (Figs. 1 and 2). Dried polyvinyl emulsions are not truly waterproof. W h e n they are resoaked they soon lose their translucency, b e c o m e opaquely white, swell, and tend to disintegrate. During sectioning this relatively slow process is not a serious limitation with solid epithelial tissues which do not permit appreciable glue penetration into their m a s s . But it does p r e s e n t a problem with tissues s u c h as lung, with its large cavities to be filled (Figs. 1 and 4), and also where natural surfaces m a y need support as with ciliated tracheal epithelium (Fig. 5) or intestinal b r u s h border (Figs. 8 and 9). We discovered, however, that if we added a small a m o u n t of s e r u m albumin to the glue before drying it, and then later exposed the dried sample to 2% glutaraldehyde, the albumin p r e s u m a b l y cross-linked it, preventing the tendency for the glue to fall apart w h e n wet. It takes only one drop of a 30% s e r u m albumin solution per milliliter of glue to achieve this. (A word of caution is necessary. If an a t t e m p t is m a d e to add appreciably more albumin to the glue than is suggested, a p h a s e separation or precipitation occurs which then renders the emulsion useless. On the other h a n d , at least small quantities of glue, with the proper a m o u n t of s e r u m albumin added, s e e m to be indefinitely stable.) H o w tissue can be supported m o s t effectively dep e n d s partly on the nature of the specimen. In any case, the tissue is trimmed in a d v a n c e to produce blocks not m u c h larger than their final size. It is worth repeating that e x c e s s fixative always is then thoroughly w a s h e d from the tissue blocks by passing t h e m through changes of a neutralized physiological saline solution. W a s h e d s p e c i m e n s are i m m e r s e d in about 2 ml of glue (with the albumin already added if it is deemed desirable), and then slowly tumbled. T h e tumbling ordinarily is continued for about 30 to 60 min, and longer in the case of a tissue such as lung, to be sure that the alveolar spaces (or other cavities) are completely filled with the glue. To be sure, the prolonged mixing probably is not ordinarily necessary, particularly with a d e n s e epithelial tissue such as kidney, liver, or pancreas. We have successfully prepared such s p e c i m e n s with only a few minutes of stirring. There m a y be, however, a secondary advantage to a s o m e w h a t long exposure to the emulsion in that no doubt a partial, and relatively slow, dehydration of the tissue occurs at that time under relatively controlled circumstances. T h e next step after infiltration involves removing individual blocks from the bulk of the glue, generally with a toothpick, and placing each block on a polyethylene sheet with j u s t e n o u g h e x c e s s glue to maintain a coating layer as the block dries. If desirable, the tissue blocks are oriented at this time. In the course of j u s t a few m i n u t e s , the glue loses its opaque whiteness and b e c o m e s translucent as it dries. T h u s , the s p e c i m e n s finally are easily seen.

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Overnight drying is all that is really n e e d e d before proceeding with the next step. In practice, h o w e v e r , we have generally u s e d a v a c u u m o v e n operating at 37°C, with a negative p r e s s u r e corresponding to 10-15 m m Hg, to thoroughly dry the blocks. After the glue is dried, if it contains s e r u m albumin, the s p e c i m e n s then are resoaked for a few minutes in 2% glutarald e h y d e to achieve cross-linking. T h e glue quickly bec o m e s opaquely white as it is rewet, b u t the crosslinked albumin p r e v e n t s it from swelling appreciably or disintegrating. The blocks then are r e w a s h e d and redried. B e c a u s e ordinarily a n u m b e r of s p e c i m e n s will be arranged on a single polyethylene sheet, and will remain fastened to the sheet during t h e s e processing steps, it is only n e c e s s a r y to r e m o v e the sheet from the one bath to another, and finally redry this. Selected s p e c i m e n s finally are glued to supporting stubs that will fit the collet of the microtome. Generally, these are cut from suitably sized wooden dowel rods, although old plastic m o u n t s can be u s e d as well. (Glues s u c h as epoxies which might have an organic solvent action are avoided in specimen mounting.) Tissue blocks are t r i m m e d conventionally, perhaps slightly smaller than usual.

Sectioning Ultrathin sectioning is surprisingly easy if certain guidelines are strictly followed. We feel that glass knives should be u s e d rather than diamond ones in spite of c o m m e n t s in the literature implying the contrary. It is not a question of relative s h a r p n e s s , b u t rather how the trough fluid wets the knife edge. It is particularly glass that will support a sufficiently low water m e n i s c u s so that the block face is not too apt to be wet as sections are obtained. T h e minimal meniscus desired can hardly be obtained with water w h e n using an ordinary trough. Instead, control of the meniscus is m o s t readily realized if the fluid is contained only by a band of hydrophobic fingernail polish (or wax), applied as a water barrier, about 5 m m below the knife edge. The clearance angle of the glass knife is set at 3 ° . W h e n facing a n e w block with a dry knife, a jet of c o m p r e s s e d Freon or air is the best way of removing debris. After serious sectioning is begun, with water in the trough, debris at the knife edge can be r e m o v e d safely with a hair or by drawing a wooden spatula made from a toothpick, along its edge, as one does with a diamond knife. This last technique is also a useful m a n e u v e r in helping to keep the knife wet to its very edge. M o s t of our experience to date has been with pure water as the trough fluid, but recently we have c o m e to realize that ethylene glycol, u s e d either undiluted or mixed with water, m a y offer substantial advantages. T h e surface tension of pure ethylene glycol is about two-thirds that of water (48 d y n e s / c m as corn-

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pared with 73). Its viscosity at 20° is 21 cp, compared with 1 cp. A notably higher meniscus can be mainmined without wetting the block face, even with only a 10% solution of ethylene glycol. An advantage of the reduced surface tension of glycol mixtures is that nonhomogenous tissues are less apt to be pulled apart as they flatten on the fluid surface. There appears also to be a damping effect during sectioning so that cutting artifacts are lessened. It is often possible with glycol mixtures to reduce the cutting speed substantially. Even pure ethylene glycol is regarded as being so polar that it is doubtful that the physicochemical properties of tissue are much modified by its use in preference to water. The only practical disadvantage that we are aware of so far is the difficulty encountered when a block is wet by a glycol solution, for the fluid will not simply evaporate as will pure water. Although a few thick sections may remove a limited wet layer, and thus permit resumption of ultrathin sectioning, in severe situations wet blocks may have to be flushed with water, and then redried. Our own sectioning is done largely with a Sorvail MT-2 or MT-1 microtome. In general, however, we do not use a motor drive, primarily because hand-turning provides much greater flexibility, permitting one or two initial sections to be taken rapidly, and then if all seems to be going well, reducing the cutting speed. A speed of about 1.5 to 2.5 mm/sec generally seems optimal with pure water in the trough. When tissue has been fixed only with glutaraldehyde, it generally is not practical to section at a nominal thickness of less than 800/~. On the other hand, material secondarily fixed with osmium tetroxide and/or tannic acid, and/or uranyl salts, permits substantially thinner sectioning (400-500/~). Sections generally are mounted on grids with supporting films. These are not strictly necessary, however. Although tears have to be expected to occur in unsupported sections, areas of section which partly stretch across 400-mesh grid squares often can be stabilized by an initial exposure to low levels of electron bombardment, and then small areas can be profitably examined at high magnification with an intense beam. Staining

A major problem that has only partly been resolved is how best to stain unembedded sections. The one quite effective, but unfortunately limited, method so far available is the use of partially neutralized phosphotungstic acid (PTA), as Farrant and McLean (1969) originally did in studying isolated organelles and microorganisms supported by cross-linked albumin, and as used by Nicolson (1971) who applied the same technique to study isolated chloroplasts. There is no question but that to a considerable extent PTA acts as a negative stain, but it is hard to know to what extent it may also act positively. The net effect is that membranes are seen in negative contrast. Other parts of

cells and tissue components are apt to be rather uniformly stained, an exception is elastica which remains unstained, as sometimes does the concentrated hemoglobin of nonhemolysed erythrocytes (Fig. 6). The pattern of PTA-stained collagen, however, is that of a positive stain (Fig. 12). In using PTA as a stain, 2% solutions at pH 5.0-6.5 can give adequate contrast to unsupported sections. Stronger solutions (5 or 10%) are used with supported sections. Grids are inverted on the surface of drops of stain on a sheet of dental wax. Staining time is usually 2-5 rain, and then the grids are blotted between a folded sheet of filter paper without washing. The vagaries of negative stains in particular are notorious, and we cannot claim a uniform product under the best of circumstances. One source of difficulty relates to inconsistent wetting. A trace of "Fotoflo" in the stain sometimes seems to be helpful. The PTA stains are reasonably stable, but precipitates may form so that it is our practice to inspect bottles frequently and filter when necessary. Five Percent uranyl acetate has also been used as a stain. It behaves much like PTA, generally as a negative stain, but exhibits less contrast. There is a slight positive staining effect on nucleoproteins, but by itself this is not enough to be particularly useful. The uranyl oxalate stain of Tokuyasu (1978), used at approximately neutral pH, has not seemed an improvement. A 5% aqueous solution of uranyl acetate, used en bloc for relatively brief periods ( - 1-3 hr), does not importantly help contrast, although it does improve the sectioning potential of the treated tissue. Prolonged (overnight) en bloc treatment of tissue with 0.5% uranyl magnesium acetate behaves in much the same way, improving sectioning and producing a barely perceptible contrast enhancement of nucleoproteins, as well as some fibrous proteins such as myosin. Subsequent treatment of the sections with partially neutralized PTA, however, then does result in a useful reinforcement of the nucleoprotein stain. A considerable effort has been made to use OsO4 as a section stain; either alone, or following other treatments. We have had singularly little success no matter whether sections were floated on OsO4 solutions, or exposed to OsO4 vapors in small closed containers. In the latter case, both wet and dry sections have been given either brief (3- to 6-min) or long (30- to 60-min) exposures. Mild heat has been used to increase OsO4 vapor pressure. In fact, sections have even been immersed in pure, molten OsO4 (<50°C). Both the OsTMEDA tetramethylethylenediamine stain introduced by Seligman et al. (1968), and the ferrocyanide-reduced OsO4 stain of Karnovsky (1971) have been used as well without notable success. Other stains which have been employed include Reynold's lead citrate, the vanadyl sulfate and vanadatomolybdate stains of Callahan and Horner (1964), the Riva (1974) technique of using bismuth subnitrate

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SECTIONING UNEMBEDDED TISSUES following uranyl acetate treatment, and the ruthenium red-osmium tetroxide procedure of Luft (1971). We have also employed the lead asparate stain en bloc as developed by Walton (1979) and additionally have attempted direct staining of sections with it. None of these procedures, however, have been significantly successful in enhancing the differentiation of unosmicated tissue. In using ferritin-labeled material we have successfully used a bismuth subnitrate technique as advocated by Ainsworth and Karnovsky (1972) to enhance the size of the label. Since this involves a 30- to 60-min exposure of the sections to the highly alkaline (pH -12) stain, we take the precaution of refixing such sections for 10 to 20 min with 5% glutaraldehyde (with subsequent washing) as a prelude to the staining procedure. This prevents any morphological deterioration of the specimens. Lectin and Antibody Binding A successful experiment has been performed in collaboration with Dr. Izhak Nir of the Technion School of Medicine, Haifa, Israel, specifically binding ferritinlabeled concanavalin A to the glycogen-rich paraboloid region of frog retinal cones (Fig. 22). The special technique and the materials employed have been published in detail by Nit (1978). Also, a successful pilot experiment involving the localization of antirod outer segment antiserum with ferritin-labeled IgG in the frog retina has been presented and published in abstract form (Pease et al., 1981). The detailed report has been submitted for publication (Pease et al., 1982). Shrinkage Artifact It seemed desirable to have an estimate of the shrinkage that might occur in infiltrating and drying tissue specimens in the manner reported here. Therefore, the lengths of 25, rectangularly shaped, small blocks; both of liver, and of kidney cortex, fixed only in glutaraldehyde, were carefully measured while still in

fixative, and finally again after 4 hr of tumbling in white glue, and drying the specimens in our usual manner. The linear measurements were made quite accurately at a 20-fold magnification with a "Nikon 6C Profile Projector," equipped with a micrometer stage. The liver blocks demonstrated a 23% shortening (range 21-26%), and the kidney specimens, 25% (range 21-29%). The "Profile Projector" was also used to measure myelin periodicity with accuracy. RESULTS Sectioning Characteristics and Artifacts The illustrations included here demons t r a t e t h a t it is p o s s i b l e to o b t a i n u n i f o r m and adequately thin sections of a n u m b e r o f cell t y p e s an d o r g a n e l l e s e v e n t h o u g h t h e y h a v e b e e n f i x ed o n l y w i t h g l u t a r a l d e h y d e (Figs. 1 an d 4-22). It is r ar e, h o w e v e r , to o b t a i n a l ar g e c o n t i g u o u s a r e a o f sect i o n e d t i s s u e w i t h o u t o b j e c t i o n a b l e flaws. O f t e n t h e l a t t e r ar e s i m p l y u n s i g h t l y h o l e s a n d t ear s (Fig. 1). In t h e c a s e o f u n s u p p o r t e d s e c t i o n s , h o w e v e r , t h e s e t e n d to expand disastrously during electron bombardm e n t . F o l d s a l s o ar e c o m m o n (Fig. 1), an d c a n be r e s p o n s i b l e f o r an u n e v e n an d unsightly d i s t r i b u t i o n o f p h o s p h o t u n g s t i c acid stain. F u n d a m e n t a l l y m o r e s e r i o u s flaws o c c u r w h e n t h e knife d o e s n o t cu t s m o o t h ly, b u t i n s t e a d l e a v e s a r o u g h s u r f a c e . It is n o t u n c o m m o n to find r o u g h l y cu t a r e a s c l o s e b y s m o o t h o n e s , or o n e s e c t i o n m a y have been cut excellently while a neighboring s e c t i o n o n t h e s a m e grid is r u i n e d b y s u r f a c e r o u g h n e s s . C o m m o n l y s e c t i o n s ar e n o t o f u n i f o r m t h i c k n e s s . C o a r s e c h a t t e r is o n l y an o c c a s i o n a l p r o b l e m .

FIG. 1. Sectioned lung alveolar walls, supported by a dried polyvinyl-acetate emulsion. Residual, internally directed, elastic forces within the support system have thrown the tissue section into a series of folds. Other sectioning artifacts include holes. PTA stain, x 2875. Fro. 2. Dried and sectioned polyvinyl-acetate emulsion with PTA stain outlining the individual particles. The larger particles range in size up to about 5 p~m. x 7000. FIG. 3. Diluted polyvinyl-acetate emulsion, dried on a support film. Many small particles range in size down to about 0.1 ~m. x 8750. Fro. 4. Longitudinal section of a capillary in a lung alveolar wall, fixed only with glutaraldehyde and stained with PTA. The serum areas have accepted a good deal of stain while the packed hemoglobin contents of the erythrocytes have not. The basal lamina is amorphously stained. The membrane systems of endothelium and epithelium are seen in negative contrast, x 28 750. Fro. 5. Transverse section of glutaraldehyde-fixed cilia from the tracheal wall, negatively stained with PTA. The microtubular system is well defined in negative contrast, with the lumina generally filled with stain, x 53 750.

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I n relation to specific cellular and tissue c o m p o n e n t s , nuclei cut easily (Figs. 1 and 6). P a t c h e s o f r o u g h and s m o o t h endoplasmic reticulum (Figs. 7 and 16), the Golgi a p p a r a t u s (Fig. 6), m i t o c h o n d r i a (Figs. 6, 8, 13, and 16), b r u s h orders (Figs. 8-10 and 14), and cilia (Figs. 5), all section equally well. T h e h e m o g l o b i n o f e r y t h r o c y t e s sections particularly easily, p e r h a p s b e c a u s e o f its h o m o g e n e i t y (Figs. 1, 4, and 6). Bundles o f collagen also cut easily (Fig. 12); elastin p r e s e n t s no serious difficulty. Glyc o g e n m a s s e s h a v e s o m e t i m e s , but n o t alw a y s , p r e s e n t e d a p r o b l e m . It a p p e a r s s o m e t i m e s to be e x t r a c t e d (Fig. 22), b u t o t h e r times n o t (Figs. 11 and 16). T h e r e h a v e b e e n no particular difficulties with m o s t t y p e s o f granules that w e h a v e dealt w i t h , in n e u t r o p h i l s , e o s i n o p h i l s , m a s t cells, and different sorts o f l y s o s o m e s (Fig. 16). Z y m o g e n g r a n u l e s in the p a n c r e a s , h o w e v e r , illustrate a p a r t i c u l a r p r o b l e m . T h e y not only have a t e n d e n c y to shrink differentially, and thus pull a w a y f r o m their surrounding c y t o p l a s m , but t h e y s e e m to be relatively hard structures so that m a n y are c h i p p e d out o f a c i n a r cells and displaced or lost. One can find areas, h o w ever, w h e r e e v e n these granules h a v e b e e n

r e a s o n a b l y p r e s e r v e d a n d are well sectioned. While n e a r l y p e r f e c t low p o w e r microg r a p h s are rarities (Figs. 4 and 6), there is little difficulty in obtaining excellent highmagnification m i c r o g r a p h s o f limited areas. This is particularly true w h e n dealing with r e d u n d a n t s y s t e m s so that one c a n obtain highly s e l e c t e d e x a m p l e s w i t h o u t an ard u o u s and t i m e - c o n s u m i n g search. W h a t has p r o v e n to be particularly difficult to section are histological situations w h e r e there are a b r u p t c h a n g e s in the p h y s ical properties o f a d j a c e n t tissues. T h u s , efforts to obtain g o o d , full-thickness sections of the rat t r a c h e a , or intestinal wall, h a v e not so far s u c c e e d e d . T h e v a r i o u s layers tend to split a w a y f r o m o n e a n o t h e r , yet, with p e r s i s t e n c e , one c a n e x p e c t to be able to obtain well-sectioned material in a n y single o n e o f the layers. H o w the b l o c k is oriented relative to the knife edge c a n be decisive in t e r m s o f optimal results. T h u s , f o r the t r a c h e a l or intestinal epithelium, these layers p r o b a b l y s h o u l d face the knife edge and e n j o y the s u p p o r t o f the d e e p e r tissue while being sectioned. T h e d e e p e r layers, d o m i n a t e d b y c o n n e c t i v e tissue and s m o o t h m u s c l e , are b e s t s e c t i o n e d w h e n

FIG. 6. Parts of lymphocytes within a lung alveolar wall capillary, fixed only with glutaraldehyde, and stained with PTA. The negative stain outlines the cytomembrane systems of the mitochondria, a Golgi apparatus (lower right), as well as nuclear and plasma membranes. The nuclear contents are not differentiated. The concentrated hemoglobin of the part of the erythrocyte included in the upper right corner of this micrograph (rbc) has not been penetrated by the stain. × 30 000. FIc. 7. An area of rough-surfaced endoplasmic reticulum from pancreas, fixed with glutaraldehyde, and stained with PTA, exhibits only faint traces of ribosomes. × 190 000. FIG. 8. Intestinal epithelium, fixed only with glntaraldehyde, and stained with PTA. Much fine structure has been preserved as the cytomembrane systems outlined in negative contrast demonstrate. × 31 300. FIG. 9. The filamentous system of the intestinal brush border, and the zone immediately below it, is not differentiated in material fixed and stained comparably with that of Fig. 8 (compare Fig. 14). z 45 000. FIG. 10. Attempts to stain aldehyde-fixed tissue with alkaline lead stains have proven to be entirely unsatisfactory at high magnification because of the granularity that invariably is induced. Otherwise, the material of this figure is comparable with that of Fig. 9. × 45 000. FtG. 11. Glutaraldehyde-fixed, and PTA-stained, striated muscle. Here the glycogen particles have been heavily stained, but this has been an inconsistent finding (see also liver and retinal cone paraboloid glycogen, Figs. 16 and 22). The filamentous systems of the muscle are seen only vaguely, but there are suggestions of very well-preserved order. Conventional designations are used to indicate band areas, x 45 000. FIG. 12. A bundle of collagen from glutaraldehyde-fixed lung alveolar wall, stained with PTA. The banded pattern corresponds in this case to the typical pattern of a conventional positive stain. × 95 000.

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oriented at right angles to the knife edge. Retina, particularly the layer of rod and cone outer segments, seems to section best when the latter elements are parallel to the knife edge (Figs. 1%21). Efforts h a v e been made to completely encase fully trimmed blocks with a wrapping of glue. Thus, as sections are cut, they would be s u r r o u n d e d b y a ring of glue which might help hold t h e m together. This is effective to a limited extent only. At least a part of the p r o b l e m is that dried glue on the surface of a block c o m p r e s s e s the encased material. Sections that are surrounded by glue are apt to be more folded than otherwise might be the case. This is particularly well shown in lung tissue in which the glue fills the alveolar spaces. Although the glue m a y be sectioned v e r y smoothly, the alveolar walls invariably d e m o n s t r a t e fairly regularly and closely spaced folds (Fig. 1). In sectioning natural flat epithelial surfaces, such as the lining of the gut wall (Figs. 8 and 9) or of the trachea (Fig. 5), h o w e v e r , a surface layer of glue cannot be avoided. Also, v e r y small objects including retina m a y have to be encased in glue to be cut transversely (Fig. 19-21). These are tolerable practices even though it is generally a d v a n t a g e o u s to trim a w a y all glue when possible at the edges of a block of solid tissue. If one is willing to use o s m i u m tetroxide as a s e c o n d a r y fixative, then the potential for sectioning u n e m b e d d e d tissue is substantially enhanced. As indicated under Materials and Methods, it is rarely practical to attempt the ultrathin sectioning of tissue fixed only in glutaraldehyde at less than 800

thickness. It is c o m m o n l y possible, however, to reduce section thickness to 400500 A after osmium-tetroxide fixation. Furthermore, one can expect to find m u c h larger areas of r e a s o n a b l y u n i f o r m sections without serious artifacts. Thus, relatively l o w - p o w e r micrographs are c o m m o n l y possible. In this regard, a-postglutaraldehyde treatment with uranyl acetate or dilute uranyl m a g n e s i u m acetate also p r o v e s to be quite helpful, e v e n without p o s t o s m i c a tion, e m p h a s i z i n g that t h e s e s u b s t a n c e s must indeed h a v e some of the properties of true fixatives. We were particularly interested in gaining insight as to h o w lipids might be preserved and sectioned without e m b e d m e n t s , so that a fairly extensive study of peripheral-nerve myelin was undertaken. N o t surprisingly, thick sections of nerve fixed only in glutaraldehyde d e m o n s t r a t e a strong birefringence w h e n examined with a polarizing m i c r o s c o p e . T h e r e is always pattern irr e g u l a r i t y a p p a r e n t in s u c h s e c t i o n s , however. In the a b s e n c e of an e m b e d m e n t , w h e n glutaraldehyde is the only fixative, thick layers of myelin invariably are fragmented. One obtains flakes that in limited areas m a y be well p r e s e r v e d (Figs. 17 and 18). L o c a l regions of s m e a r e d m e m b r a n e s must also be e x p e c t e d as in the lower left and right areas of Fig. 17. On the other hand, after a s e c o n d a r y fixation with o s m i u m tetroxide (sometimes with tannic acid and/or uranyl salts), the myelin periodicity survives sectioning quite beautifully, in m o s t places without disturbance. Other stacked b i o m e m b r a n e s y s t e m s are

FIG. 13. Kidney distal tubule, glutaraldehyde fixed and PTA stained. Preservation has been generally good although the somewhat uneven spacing between the paired infolded plasma membranes indicates a degree of collapse of fine structure. Even at high magnification, differential detail is not evident in the inner mitochondrial membranes (compare Fig. 16). × 60 000. FIG. 14. Brush border of the proximal tubule of kidney, prepared as the tissue of Fig. 13. No internal filamentous structures are to be seen although the overall preservation of the brush border has been good (compare Fig. 9). × 55 000. FIG. 15. A desmosome from tracheal epithelium, fixed only in glutaraldehyde and stained with PTA. Although membrane components are well seen in negative contrast, tonofilaments are not evident (compare with muscle filaments, Fig. I 1). × 95 000.

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sectionable with less damage than myelin. The myeloid bodies of frog retina are part i c u l a r l y s u c c e s s f u l l y c u t (Fig. 20), as a r e r e t i n a l c o n e o u t e r s e g m e n t s (Fig. 21). R o d outer segments show greater perturbations (Fig. 19), b u t it s e e m s l i k e l y t h a t this r e p resents properties of their preservation r a t h e r t h a n p r o b l e m s with s e c t i o n i n g . It is a n i n t e r e s t i n g f a c t t h a t r e l a t i v e l y solid e p i t h e l i a l t i s s u e s e x h i b i t a s u b s t a n t i a l h y d r o p h o b i c i t y . T h a t is to s a y , as s e c t i o n s float on a w a t e r s u r f a c e , t h e i r o w n s u r f a c e r e f l e c t s light t h a t is s i l v e r , g o l d , o r c o l o r e d , depending upon their thickness. Unless a b l o c k h a s b e e n w e t , t h e r e is n o t m u c h o f a t e n d e n c y f o r t h e s e c t i o n s to s i n k . O n t h e other hand, blocks made up principally of c o n n e c t i v e t i s s u e y i e l d s e c t i o n s t h a t a r e imm e d i a t e l y p e n e t r a t e d b y t h e w a t e r so t h a t they do not demonstrate good reflecting surfaces. Floating sections then can be q u i t e difficult e v e n to see. S u c h b l o c k s t h e m s e l v e s a l s o a r e p a r t i c u l a r l y p r o n e to b e i n g w e t . I n t h e s e r e s p e c t s , w e h a v e exp e r i e n c e d t h e g r e a t e s t difficulty with r a t u t e r u s , b u t h a v e a l s o h a d p r o b l e m s w i t h the layers of the intestine and trachea dominated by connective tissues. Long ribbons of sections are not usually

o b t a i n e d f o r t h e r e is little t e n d e n c y f o r ind i v i d u a l s e c t i o n s to a d h e r e to o n e a n o t h e r . C o m m o n l y , rafts o f i s o l a t e d s e c t i o n s a r e t h e r e s u l t . A c o r o l l a r y o f this is t h a t b l o c k faces of quite irregular shape often can be cut successfully. S e m i t h i c k s e c t i o n s f o r light m i c r o s c o p y are obtained easily, and transferred to slides f r o m t h e w a t e r s u r f a c e w i t h a p l a s t i c loop. They stain brilliantly with conventional dyes. Aqueous dyes and mounting m e d i a c a n b e c h o s e n to a v o i d o r g a n i c solv e n t a c t i o n . D r y s e c t i o n s e v e n c a n b e obt a i n e d in t h e 1- to 3-/xm r a n g e o f t h i c k n e s s a n d t r a n s f e r r e d to a slide w i t h fine f o r c e p s o r a hair. T h e y will a d h e r e to c l e a n g l a s s a n d b e f l a t t e n e d if p r e s s e d d o w n w i t h a p l a s t i c film. S p e c i m e n c h a r a c t e r a n d b l o c k size d e t e r m i n e t h e i r o p t i m a l t h i c k n e s s . I f a t t e m p t s a r e m a d e to o b t a i n t o o - t h i n sect i o n s , t h e y will b e r u m p l e d . T o o - t h i c k sect i o n s will curl e x c e s s i v e l y . T o a v o i d c u r l i n g it is s o m e t i m e s n e c e s s a r y to h01d d o w n e a c h s e c t i o n as it is b e i n g c u t w i t h t h e tip of a hair or a plastic point. The dried polyvinyl-acetate emulsion t h a t w e h a v e r e l i e d u p o n is u n d o u b t e d l y s o f t e r t h a n is u l t i m a t e l y d e s i r a b l e , p a r t i c u l a r l y w h e n s m a l l o b j e c t s h a v e to b e fully

FIG. 16. An area of liver rich in glycogen and smooth endoplasmic reticulum is seen here after glutaraldehyde fixation and PTA staining. Also evident are two lysosomes. Although not well seen here, the latter commonly demonstrate in negative contrast ordered arrays of membranous material. As indicated in relation to Figs. 11 and 22, glycogen staining is capricious. × 75 000. FIG. 17. Peripheral nerve myelin, fixed only with glutaraldehyde, and stained with PTA. As the text indicates, only flakes of sectioned myelin are obtainable after this preservation. Regions where the intrinsic order has been smeared are commonplace, as in the lower right and left corners of this figure. × 153 700. FIG. 18. A high resolution micrograph of myelin, prepared as that of Fig. 17. It is the "intraperiod" lines, the extensions of the mesaxon, that are the conspicuous bands of these preparations. The "major" lines often are not seen at all, or at best only faintly, as in the case here. The periodicity of this preparation approximates 150 ]k. x 240 000. FIG. 19. A frog retinal rod outer segment, lightly and briefly fixed with glutaraldehyde-formaldehyde, stained with PTA. Although the disc membranes are not preserved in perfectly ordered arrays, their basic fine structural characteristics are retained and seen. × 127 500. FIG. 20. Part of a myeloid body and the smooth endoplasmic reticulum from a pigment cell of frog retina, preserved as the material of Fig. 19. × 82 500. FIG. 21. A frog retinal cone outer segment, fixed as the material of Figs. 19 and 20. During processing cone disc membranes generally maintain more precisely ordered arrays than do rod membranes. This particular retinal section had been incubated with ferritin-labeled, concanavalin A, as had the material of Fig. 22. It serves as an intrinsic control for the latter since in this area, in the absence of glycogen, no binding is evident. x 127 500.

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encased in it for support. An intrinsically harder plastic would be an i m p r o v e m e n t , and surely could be found or developed.

Staining It is unfortunate that a n u m b e r of conventional staining p r o c e d u r e s do not significantly differentiate structural elements in tissue fixed only with glutaraldehyde. The m o s t generally effective stain used to date for our material has been partially neutralized phosphotungstic acid (Figs. 1,4-9, and 11-22). It is evident, however, that almost all hydrophilic parts of such preparations stain nearly uniformly so that the value of the stain is chiefly to delineate h y d r o p h o b i c elements in negative contrast. These particularly include bilayered c y t o m e m b r a n e systems as well as accumulations of ordered lipids (Figs. 17-21). Collagen provides a unique opportunity to d e m o n s t r a t e that partially neutralized P T A can also act as a positive protein stain (Fig. 12). Elastin remains unstained, as v e r y nearly does hemoglobin (Figs. 6 and 4) in unlysed red cells. E v e n such well-organized fibrillar p r o t e i n s as myosin in skeletal muscle (Fig. 11) and the tonofibrils of d e s m o s o m e s (Fig. 15) are at best barely detectable. Microtubules are an exception, and are seen in negative contrast with their l u m i n a c o m m o n l y filled with stain (Fig. 5). Uranyl acetate, used alone as a negative stain, b e h a v e s m u c h as PTA, but without exhibiting as m u c h contrast. The interpretation of the a p p a r e n t erratic staining of glycogen presents special problems. At times it has the a p p e a r a n c e of a positive stain (Figs. 11 and 16). At other times the staining is at best faint (Fig. 22).

In liver and muscle it is sometimes quite possible to see these variations within a single grid square. It is possible that these v a r i a t i o n s are m o r e a reflection of the a m o u n t of glycogen extracted than real positive staining differences. Or, it m a y be that when high density is seen the P T A is simply filling cavities left by extracted glycogen, and so is acting as a negative stain. A t t e m p t s to stain glutaraldehyde-fixed tissue with lead citrate, either by itself, or after p r e t r e a t m e n t with uranyl salts, h a v e had only limited success. At relatively low magnifications some differentiation, particularly of nucleoproteins, can be obtained. Invariably at high magnification, though, the stain is seen to be seriously granular (Fig. 10, c o m p a r e with Fig. 9). Additionally, there often is obvious evidence of the destruction of cytological details. We suspect that m u c h of this damage is attributable to the e x t r e m e alkalinity of this stain. Reducing the standard concentration of the stain 10-fold does not fundamentally imp r o v e the situation. W h e n tissue that has been secondarily fixed in OsO4 is stained with lead citrate, the contrast differential m a y be improved. But, the granularity of the stain again destroys its usefulness for high resolution studies. As indicated under Materials and Methods, a rather wide variety of other staining techniques h a v e b e e n a t t e m p t e d without notable success on tissue fixed only with glutaraldehyde. N o doubt some of these varied p r o c e d u r e s would be effective if the i n v e s t i g a t o r would be willing to a c c e p t postfixation with OsO4. F r o m a purely mor-

FIG. 22. The insert shows part of the paraboloid (par), and the ellipsoid (el), of a cone from a frog retina, lightly and briefly fixed with glutaraldehyde-formaldehyde. The paraboloid is known to contain glycogen. In this case it was either mostly extracted, or only lightly stained with PTA (unlike the reactions evident in Figs. 11 and 16). This section was incubated with ferritin-labeled, concanavalin A. At high magnification the junctional region, indicated in the insert, shows a rich deposition of label over the paraboloid which does indicate that at least some glycogen was preserved throughout the treatment stages. This section also demonstrates that there was essentially no nonspecifically bound ferritin Con A on the ellipsoid, which thus serves as an intrinsic control (as does the cone outer segment section illustrated in Fig. 21 which had been similarly incubated without evidence of specific binding). (Work in collaboration with Dr. Izhak Nit, as indicated in the text.) x 215 000.

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phological point of view, however, the present lack of a differentially selective positive stain remains a major limitation. O b s e r v a t i o n s on T i s s u e s

The most surprising generalization that can be made about sections of unembedded material is that an extraordinary amount of fine structural detail is preserved by air drying after only a' minimal glutaraldehyde fixation. Thus, it is evident that the gross structure of cells is not seriously distorted, in spite of some overall shrinkage, so that their more delicate features are well seen. These include brush borders (Figs. 8, 9, and 14), cilia (Fig. 5), and infolded plasma membranes (Fig. 13). In general, cytomembrane systems are well preserved and are easily observed in negative contrast, outlining membrane-bound intracellular compartments of all sorts, including e.g., the triads of skeletal muscle (Fig. 11). It is unfortunate that hydrophilic protein systems, including filamentous ones, are largely masked for want of staining systems which will permit their differentiation. When they can be seen, as in microtubular systems (Fig. 5), collagen (Fig. 12), and to a very limited extent, as the myosin filaments of skeletal muscle (Fig. 11), it is evident that they too are intrinsically well preserved. Since the technique permits the retention and observation of lipids that are insoluble in water, and those that may be immobilized by glutaraldehyde, it has been a disa p p o i n t m e n t that partially neutralized phosphotungstic acid, acting as a negative stain, has not revealed more cytomembrane details, particularly in such loci as the inner membrane system of mitochondria (F.igs. 13 and 16). Also, we had hoped to see more of the unit structures of ordered proteins as in microtubules (Fig. 5) and contractile systems (Fig. 11). Some deliberate efforts have been made to differentially extract cytosol prote!ns in an effort to unmask fibriUar systems. These efforts have largely been limited to striated muscle, and have been partly successful in

that if sections are exposed to" agents such as are used in ordinary dehydration procedures (alcohol, acetone, propylene oxide, etc.) components are removed. But without the benefit of a supporting embedment, what remains tends to collapse or condense upon surviving elements so that obvious artifacts attributable to surface tension and adhesion are the end results. Conceivably, extracted sections which could be kept wet, subsequently might be frozen dried or critical point dried with better success, but this has not so far been attempted. Nerve myelin has presented a particular challenge. Individual flakes of myelin, which are fixed only with glutaraldehyde, and which are well sectioned, demonstrate a uniform periodicity. It is the intraperiod line, the continuation of the mesaxon, that is prominently stained, presumably because it represents an originally aqueous, hydrophilic compartment. The " m a j o r " line often is not seen at all, or at best rather faintly (Figs. 17 and 18). While thick sections of glutaraldehyde-fixed myelin exhibit an intense birefringence, the periodicity seen in thin sections averages only 150 ,~. Furthermore, there is an unusually wide range of variation of periodicity from one sample to another, from about 120 to 165 ,~. We believe this represents compression or stretching during the sectioning of what must be a fairly plastic system. This is also born out by a frequent observation that in small regions the prominent lines may obviously converge or diverge which surely is a result of such distortions. Smearing and distortion of the myelin lamellae is largely prevented if the material is postfixed with osmium tetroxide, with or without additional fixation by tannic acid and/or uranyl salts. A major goal of the present work has been to develop a method of preparing ultrathin sections that will permit the exposure of their intracellular contents to macromolecular labels, particularly antibody molecules. Pilot experiments have demonstrated the feasibility of this in one system,

SECTIONING U N E M B E D D E D TISSUES

incubating frog retinal sections with rabbit retinal antirod serum. The preparations subsequently were successfully labeled with antirabbit, ferritin-conjugated IgG (Pease e t al., 1981, 1982). Additionally, in other experiments performed in collaboration with Dr. Izhak Nir, it has been possible to show ferritinlabeled concanavalin A binding to the glycogen-rich paraboloid region of frog retinal cones (Fig. 22). This figure also shows the adjacent, mitochondrial-rich, ellipsoid area to be essentially without binding. Cone outer segments also were without specific binding (Fig. 21). Indeed, in other parts of the retina, including the rods, nonspecific binding was minimal, constituting an intrinsic control for the specific localization of sugar residues in the paraboloid. DISCUSSION

The capability of making ultrathin sections of unembedded tissue, fixed only in glutaraldehyde, has demonstrated that a great deal of fine structure is preserved, in spite of tissue blocks being air dried. An apparent reason for success is that glutaraldehyde cross-links cytosol and other proteins to such an extent that essentially all macromolecular cellular components are rendered immobile, and the protein contents of the cells themselves then provide sufficient support for sectioning. This has been clearly stated by Griffiths and Jockusch (1980) in relation to using cross-linked serum albumin as the supporting agent. Unfortunately, the surviving matrix of cytosol proteins effectively masks other components which so far largely have not been amenable to desirable differentiation by the staining techniques that have been attempted. A corollary of this is that in life there must be substantial quantities of cytosol proteins that are ordinarily lost in conventional preparative procedures. The appearance of cytoplasm and its components in the present experiments agrees well with that seen in certain other

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types of preparations which also succeed in avoiding organic solvents and conventional dehydration procedures, as well as an OsO4 fixation. Thus, the micrographs presented here particularly resemble those that emanated from the Paris laboratory of Bernhard between 1964 and 1971 when his group was especially developing frozen sectioning (references cited under introduction). They also resemble the more recent micrographs of frozen-sectioned tissues by Tokuyasu (1973, 1978, 1980), and by those who have extended his techniques. They are similar in character to the "embedments" of Farrant and McLean (1969), and their successors, including Nicolson (1971), Kraehenbuhl e t al. (1976, 1977), Papermaster et al. (1978a,b), and Griffiths and J o c k u s c h (1980), who have all used cross-linked albumin gels for support. Furthermore, they are even remarkably like micrographs of tissue embedded in glutaraldehyde polymerized with urea in the presence of substantial quantities of water (Pease and Peterson, 1972). The common feature of all of these procedures is the preservation of most, if not all, of the macromolecular contents of the cytosol. The chief value of the present method is thought to lie in its potential for immunocytochemical applications. We know that tissue fixed minimally and only in aldehydes retains much antigenic specificity. The present procedure obviously is a technique which exposes cell contents, rendering them capable of reacting with agents that otherwise would not enter intact cells. That, plus the fact that some globular cytosol proteins are enzymes that are not membrane bound, and might well be diminished or lost in conventional procedures, provide attractive possibilities for future work. In a very real sense, this method offers a logical alternative to ultracryomicrotomy. It has the capability of reducing the amount of glutaraldehyde fixation demanded by the cross-linked serum albumin support inherent in the Farrant and McLean (1969) method. It, of course, avoids the use

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of potentially disruptive detergents or ice crystal deformation for opening cells to permit the penetration of iarge molecules. Clearly, it represents an alternate method for immunocytochemical investigations which possesses some distinct advantages. This work has been supported in part by National Science Foundation Grant PCM 78-09596 and National Institutes of Health Grant HL-01770. REFERENCES AINSWORTH, S. K., AND KARNOVSKY, M. J. (/972) J. Histochem. Cytochem. 20, 225-229. BERNHARD, W., AND LEDUC, E. H. (1967) J. Cell Biol. 34, 757-786. BERNHARD, W., AND NANCY, M. T. (1964) J. Microsc. 3, 579-588. BERNHARD, W., AND VIRON, A. (1971) J. Cell Biol. 49, 731-746. BORYSKO, E. (1963) Microsc. Cryst. Front 14, 7-13. CALLAHAN, W. P., AND HORNER, J. A. (1964) J. Cell Biol. 20, 350-356. CHRISTENSEN, A. K. (1971) J. Cell Biol. 51, 772-804. FARRANT, J. L., AND MCLEAN, J. D. (1969) Proc. Electron Microsc. Soc. Amer. 27, 422-423. FERNANDEZ-MORAN, H. (1950) Exp. CellRes. 1, 30% 340. FERNANDEZ-MORAN, H. (1952) Ark. Fys. 4, 471--484. FERNANDEZ-MORAN, H., AND FINEAN, J. B. (1957) J. Biophys. Biochem. Cytol. 3, 725-748. FULLAM, E. F., AND GESSLER, A. E. (1946) Rev. Sci. Instrum. 17, 23-35. GILEV, V. P. (1956) in SJOSTRAND, F. S., AND RHODIN, J. (Eds.), Electron Microscopy, Proc. Stockholm Conf., pp. 113-114, Almqvist & Wiksell, Stockholm. GIL~V, V. P. (1958) J. Ultrastruct. Res. 1,349-358. GOSSELIN, R. E., HODGE, H. C., SMITH, R. P., AND GLEASON, M. N. (1976) in Clinical Toxicology of Commercial Products, 4th ed., p. 788, Williams & Wilkins, Baltimore.

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