Effect of glutaraldehyde and urea embedding on intracellular ionic elements

Printed in Sweden Copyright © 1974 by Academic Press, Inc. All rights of reproduction in any form reserved J. U L T R A S T R U C T U R E RESEARCH

49, 495-418 (1974)

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Effect of Glutaraldehyde and Urea Embedding on Intracellular Ionic Elements X-ray Microanalysis of Skeletal Muscle and Myocardium R. YAROM,P. D. PETERS,and T. A. HALL

Department of Pathology, Hebrew University-Hadassah Medical School, Jerusalem, Israel and Electron-Microscopy Section, Cavendish Laboratory, Cambridge, England Received January 14, 1974 Embedding without dehydration in a polymerizable mixture of glutataldehyde and urea was tested o n skeletal muscle and myocardium prepared in various ways. This method, which theoretically should decrease electrolyte disturbances, appears preferable to conventional techniques for electron microscope X-ray microanalysis and histochemical studies. Analysis of minimally treated tissues showed that chlorine and calcium are easily detectable as intraceltular elements when precipitated with silver and antimony, respectively. Glutaraldehyde + urea embedding proved visually and analytically superior to Epon in preserving ionic stability. An experimentally produced increase in intracellular myocardial calcium was also better reflected by this method. Several points of interest relating to nuclear calcium shifts in the myocardium were noted. Electron microscopic demonstration of changes in intracellular ionic distribution combined with " o n the spot" checking by X-ray microprobe analysis represents an invaluable method for correlating structure and function in physiological and pathological processes. The main difficulties in this combined method are the elaborate tissue preparation techniques which may introduce many artifacts and standardization problems. Even quick freezing and ultracryotomy cannot be relied upon to preserve and present in vivo conditions (2). Without a satisfactory electron microscopic cold stage, the freezing, thawing, and drying processes may introduce many extraneous changes. In previous work (15, 16, 18) dealing mainly with the intracellular distribution of calcium in muscle and myocardium, we tried to combine histochemical and analytical techniques for morphological demonstration of calcium localisation and fluxes. However, the variability of results made evaluation and interpretation very difficult. It appeared, therefore, worthwhile to test the elimination of some steps in tissue processing, known to be associated with ionic shifts and losses (1, 3). Pretreated

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tissues were embedded in a polymerizable mixture of glutaraldehyde and urea without prior alcohol dehydration. The effects of this procedure were observed by visual and analytical means. In addition to comparison of different fixation and embedding techniques, experiments were conducted in which the ionic composition of the tissue was altered. It was hoped in this way to assess the advantages of the relatively simple and rapid embedding technique in subcellular analyticaI studies. Although preliminary in nature, the results of this study may be of interest to researchers dealing with intracellular ionic distribution and ultrastructural analytical histochemistry. MATERIALS AND METHODS

Fixing and embedding techniques. Frog sartorius muscles and rat myocardium were fixed in several different ways. These included: (a) fixation for 30 minutes in 2% glutaraldehyde buffered with 0.2 M sodium cacodylate; (b) fixation for 60 minutes i u 2 % glutaraldehyde (buffered as above) with 1% silver acetate added (9); (c) fixation for 60 minutes in 1% osmic acid and 1% sodium oxalate (buffered with 0.1 M sodium cacodylate); (d) fixation for 30 minutes with 2% potassium pyroantimonate +3 % sucrose, dissolved in 0.01 N acetic acid, O followed by postfixation for 60 minutes in either 2/o glutaraldehyde/or 1. 0Voosmic acid, both containing 1% sodium oxalate and cacodylate buffer; (e) fixation for 60 minutes in 2% potassium pyroantimonate (as in solution 4)but mixed with 1% osmic acid (8). The fixation solutions were kept on ice and the pHs were adjusted to 7.2 for the frog sartorius muscle and to 7.4 for the rat myocardium. All the samples were divided in two parts: half were rinsed in buffer, dehydrated in graded alcohols and propylene oxide, and embedded in Epo n 812; the others"were placed directly (without dehydration or other treatment) into a mixture of glutaraldehyde and urea for rapid polymerization. The principle and practical details of the embedding in the glutaraldehyde and urea mixture have been described at length by Pease arid Peterson (11, 12). Briefly, glutaraldehyde and urea are polymerizable even in the presence of large amounts of water. Lowering the pH (with oxalic acid) of a mixture of 50 % glutaraldehyde and an equal amount (on a molar basis) of urea induces the polymerization. Our procedure consisted of putting the finely chopped pretreated tissue into the acidified glutaraldehyde + urea mixture and, after a few minutes, placing each pieCe into a small drop of the polymer on a flat surface. The most satisfactory preparations were obtained when the drops took about 1-2 hours to harden. This was achieved by lowering'the pH with 0.1 N oxalic acid, to about 4.5, instead of the originally recommended pH 4 (this latter produced in our hands a too rapid polymerization and often a milky opacity which ruined the specimens). For cutting, the small hard pieces of polYmerized material containing the still-hydrated tissue were glued to an old Epon block. Although the material was very brittle and crumbled when trimmed, cutting fine sections With glass knives was unexpectedly easy when cutting speeds were higher than usual, and the results were surprisingly good. Electron microscopy and X-ray microanalysis. Sections , 100-150 nm thick, were cut onto Formvar- and carbon-coated copper grids from the Epon and the glutaraldehyde +urea embedded tissues. The sections were examined unstained in the EMMA-4 analytical microscope (AEI Scientific Apparatus Ltd., Manchester, England).

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The theory and practice of electron microscope X-ray analysis are now well known (4-6). Both the crystal diffractive and the energy dispersive (Kevex Si/Li) spectrometers were used in analyses. In the latter, elemental spectra shown on the display screen were photographed after 100-second analysis. For a more thorough study of the calcium concentrations in the differently prepared tissues, crystal diffractive analyses were done, using PET (pentaerythritol) and LiF crystals. For maximum contrast, the accelerating voltage was 40 kV. The probe diameter was approximately 200 nm, and the current was varied between 80 and 200 nA. Depending on the crystal used (PET was much more efficient), 20- or 40-second pulse counts were recorded. Readings of a standard of known composition (dentine) were taken whenever any of the instrumental conditions was changed. The relative mass fractions R presented in Table I were calculated by subtracting from the peak count P (number of pulses recorded with spectrometer in the characteristic K~I calcium position), the background reading B (spectrometer away from calcium) and dividing this P-B value by the continuum or "white" radiation W, (measure of specimen mass) after a correction was made for supporting material (WB). Thus, R -P-B)/W-WB). Dividing the R of the specimen (R~) by the R of the standard (Rs0, taken in the same way, allowed comparison between different samples and readings of different days.

RESULTS

Frog muscle fixed in different ways Frog sartorius muscles, briefly fixed with buffered 2% glutaraldehyde and then directly embedded in the glutaraldehyde + urea mixture, were fairly stable under the electron beam, but the unstained sections had very little contrast and the definition of intracellular structure and cytornembranes was poor. Nevertheless, ultrastructure was recognizable in the 120-nm sections; the nuclei were easiest to see, sarcomeres could be distinguished from the sarcoplasmic reticulum, and the Z and M lines were often discernible (Figs. 1 and 2). This allowed localization of the electron probe for X-ray analysis of various intracellular areas. The elemental spectrum of each analyzed area appearing on the display screen of the Kevex Si/Li energy dispersive analyser was photographed (Figs. 3-5). In a 100second energy dispersive analysis of this minimally treated muscle tissue, most areas proved of little analytical interest (under these instrumental conditions). What emerged from an interpretation of the displayed spectra was as follows. The small peaks between 1 and 1.5 keV were difficult to resolve. The high peak at 1.89 keV was identified as tungsten (probably due to the filament burning out during analysis). The sulfur peak at 2.31 keV was mainly due to the instrument and decreased markedly when the decontaminator was used. The two high peaks at 4.51 and 4.93 keV were from the titanium specimen holder; in higher energy ranges, only copper from the grid was registered. The sizable peaks related to the tissue itself were of chlorine at 2.62 keV and of calcium at 3.69 keV (a small potassium peak at 3.31 keV appeared 27- 741826 Y. Ultrastructure Research

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F ~ . 6. Transverse section of frog sartorius muscle, fixed in osmium and oxalate and embedded in the glutaraldehyde+ urea mixture. Nucleus (A) and cytomembranes (C) are visible, x 13 500. F1G. 7. Spectrum of area A in Fig. 6 showing the calcium peak at 3.69 keV (illuminated). F1G. 8. Spectrum of area C in Fig. 6. Calcium is present, but in lesser concentration than in the nucleus (see Fig. 7). Other areas also contained detectable calcium.

F i t . I. Unstained, 100-150 n m thick (as are all sections shown, unless specified otherwise) transverse section of frog sartorius muscle fixed briefly in glutaraldehyde and embedded in glutaraldehyde + urea mixture. The contrast is poor, but intracellular structures can be identified. The nucleus has an electron-dense marginal heterochromatiri (A) and a translucent center part (B). The sarcoplasmic reticulum (SR) separates the sarcomeres (S). x 5 000. Fie. 2. Longitudinal section of frog muscle, treated as in Fig. 1. Contrast was obtained by overexposing and overdeveloping the plate. In addition to the sarcoplasmic reticulum, myofilaments, Z lines, and M lines can be recognized. × 7 000. F~Gs. 3-5, 7, 8, and iI. Photographs of X-ray spectra in the 1-6 keV range obtained with 100-second analyses using the Kevex Si/Li analyser. FIG. 3. Spectrum of area A in Fig. 1. Only the peaks of calcium at 3.69 keV and chlorine at 2.62 keV are significant and related to the tissue. The other peaks are due mainly to supporting materials and the instrument (see text). F i t . 4. Spectrum of area B in Fig. 1, showing very little calcium. The spectrum of area A is superimposed in dots to show the difference in calcium emission. Fie. 5. Spectrum of extranuclear area S in Fig. 1 without detectable calcium. The spectra from several other sarcoplasmic locations were similar.

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in some tissues). The chlorine was present in all the areas of the minimally treated muscle; it disappeared when the probe was placed outside the myofibers. Calcium had a definite localization and was detected in the electron-dense marginal heterochromatin of the nucleus (see Fig. 3) and in the nucleolus. Calcium was not measurable in the sarcoplasm or in pale parts of the nucleus (Figs. 4 and 5). This calcium localization was also confirmed by crystal diffractive analysis (Table I, experiments 1 and 2). Muscle fixed in osmium and oxalate and embedded in glutaraldehyde + urea mixture produced slightly different results (Figs. 6-8). The contrast of the transmission image was slightly better, and the cytomembranes could be seen. In the emission spectra, no chlorine peaks appeared, whereas calcium was detected in all the intracellular areas analyzed without special loci of concentration except for the nucleus. The Epon-embedded preparations of the above tissues were not analyzed because the results proved too variable in previous work (16, 17). In addition, Epon itself contains much chlorine. These results indicated that chlorine and calcium are suitable elements for detection with X-ray microanalysis of glutaraldehyde+urea embedded tissues. Histochemical precipitation of chlorine with silver acetate and of calcium with potassium pyroantimonate was done to study the effect of varying the embedding on precipitated ions. Figures 9 and 11 show muscles fixed in 2 % glutaraldehyde to which 1% silver acetate was added, and embedded in the glutaraldehyde + urea mixture. The silver chloride was seen as a fine, uniform precipitate throughout the myofiber. The deposits often concentrated in a linear fashion in what appeared to be transverse tubules (Fig. 10). The emission spectrum with the probe over the sarcomere is shown in Fig. 11. The K~ chlorine peak at 2.62 keV is very high, and the L~ silver peak at 2.98 k e y is also prominent. Staining the grid with dry osmium vapor (for 2 hours at 30°C) gave uneven results. Some areas remained unstained and unaltered; in others the precipitate decreased. In a few cases good staining was achieved; the thick filaments, Z lines and mitochondria became especially contrasted, while the silver chloride deposits remained undisturbed (Fig. 12). When a similar specimen was rinsed, postfixed in osmium, dehydrated, and embedded in Epon, the silver chloride precipitate diminished and clumped irregularly (Fig. 13). Potassium pyroantimonate as the primary fixative and precipitator of calcium (11, 14), posffixed with either glutaraldehyde or with osmium, failed to produce any visible intracellular precipitation with either embedding material. However, m u c h nuclear and some cytoplasmic calcium were detected with X-ray microanalysis. With both kinds of postfixation, no special loci of sarcoplasmic calcium appeared; the amounts detected were small, but were higher after osmium than after glutaraldehyde. The nuclear calcium emission with these postfixations was higher than in other methods (e.g., Table I, experiment 3).

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FIG. 9. Longitudinal section of frog sartorius muscle fixed in glutaraldehyde and silver acetate, embedded in glutaraldehyde + urea mixture. A fine, evenly dispersed precipitate of silver chloride is seen inside the myofiber; Z and M lines appear free ¢f deposits. × 13 000. FIG. 10. Higher magnification of muscle, treated as in Fig. 9, showing transverse tubules containing precipitated silver chloride. × 21 500. FIG. 11. Spectrum of sarcomere in Fig. 9. High chlorine (2.62 keV) and silver (2.98 keV) peaks are present.

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T a b l e I. Relative Mass Fractions of Calcium in Various Specimens a Epon 812 Embedding Expt. No. 1 2

Specimen Fixative Frog muscle Frog muscle

Glutaraldehyde 30 minutes Glutaraldehyde + silver acetate, 60 minutes Antimonate 30 minutes, followed by glutaraldehyde + oxalate, 60 minutes Osmium + anti(7)201-+48 monate mixture, 60 minutes Os + Sb mixture, (4)121+25 as above

3

Frog muscle

4

Frog muscle

5

Rat heart, b normal control Rat heart c Os + Sb mixture after iso- as above proterenol

6

Nucleus

Glutaraldehyde + urea embedding Sarcoplasm

Nucleus

Sarcoplasm

Not analysed

(3)243-+63

None detected

Not analysed

(8)275_+48

None detected

Not analysed

(6)312__+63

(7)63_+37

(8)63_ 26

(6)227 _+32

(4) 126 -+30

(8)52_+21

(8)201_+21 (15)74_+20

Not analysed

(8)153__+22 (15)148_____43

a The values are R = (P-B)/(W-WB) normalized to one standard and multiplied by 104. Means _+SD are given. In parentheses are the number of areas analysed. b Specimens shown in Figs. 15 and 16. c Specimen shown in Fig. 17. W h e n o s m i u m m i x e d with p y r o a n t i m o n a t e served as the fixative, there was g o o d tissue p r e s e r v a t i o n a n d the typical localized calcium a n t i m o n a t e deposits m a d e electron m i c r o s c o p i c o r i e n t a t i o n easy i n - b o t h the E p o n a n d the g l u t a r a l d e h y d e + u r e a e m b e d d e d materials. The higher calcium c o n t e n t of the h y d r a t e d specimens is illustrated in T a b l e I e x p e r i m e n t 4.

Calcium localization in rat myocardium T h e a p p e a r a n c e of thicker (120 n m ) sections of r a t m y o c a r d i a fixed with p y r o a n t i m o n a t e a n d o s m i u m , e m b e d d e d c o n v e n t i o n a l l y (Fig. 15) o r in g l u t a r a l d e h y d e + u r e a mixture (Fig. 16) can be c o m p a r e d with a thin, stained E p o n - e m b e d d e d section (Fig. 14). The precipitate d i s t r i b u t i o n in m y o c a r d i u m r e s e m b l e d t h a t seen in skeletal muscle (15, 17). T h e deposits a c c u m u l a t e d in the lateral sacs a n d N lines of the sarcomeres, while b e i n g diffuse elsewhere. I n the thicker u n s t a i n e d sections, used in microanalysis, the c o n t r a s t a n d r e s o l u t i o n were p o o r , b u t the precipitate l o c a l i z a t i o n

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FIG. 12. Same specimen as in Fig. 9, stained with dry osmium vapor. Precipitates is undisturbed. × 10 500. Fro. 13. Same preparation as in Fig. 9, but with osmium posffixation, dehydration, and embedding in E~on2 The precipitate is decreased and clumped together. × 13 500.

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F~G. 14. Thin section (about 60 nm), stained briefly with uranyl acetate and lead citrate, of normal rat myocardium fixed with osmium and pyroantimonate, dehydrated, and embedded in Epon. The calcium antimonate precipitate concentrates in the lateral sacs (arrows) and along the N lines (N). x ] 9 500. FI6. 15. Same myocardium preparation as in Fig. 14, but thicker (120 rim) and unstained as used in X-ray microanalysis. The intracellular structures can be recognized and the precipitate is visible. × 18 000.

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FIG. 16. Rat myocardium fixed with osmium and pyroantimonate and embedded in glutaraldehyde + urea mixture. The precipitate appears more abundant and better delineated than in Figs. 14 and 15. x 18 000. FIo. 17. Rat myocardium injected with isoproterenol 6 hours before dissection. Specimen, fixed in osmium and pyroantimonate and embedded in glutaraldehyde+ urea mixture, shows tremendous increase in calcium antimonate, x 18 000.

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was obvious. It was much clearer and more abundant with the glutaraldehyde + urea embedding. The X-ray microanalysis (Table 1, experiment 5) correlated well with the visual differences. In experiments to increase myocardial calcium with isoproterenol, the amount of precipitate seen and analyzed increased enormously. This was much more striking in the glutaraldehyde+urea embedded material (Fig. 17). The results of crystal diffractive analysis of calcium in the osmium and pyroantimonate-fixed rat's hearts are shown in Table I, experiments 5 and 6. In addition to higher calcium emission from the hydrated tissues, the strandard error is less than in the Epon-embedded material. It may be of interest to note that while the sarcoplasmic calcium increases significantly after isoproterenol administration, the nuclear concentration of this element decreases. DISCUSSION Although glutaraldehyde +urea embedding was first used in nerves to preserve myelin lipids in situ (11, 12), the results obtained here show that it can be used to advantage in other kinds of tissues, pretreated in various ways. Especially interesting to us, in terms of our work with calcium localization and fluxes in muscle and myocardium, was the obvious increase in cellular content of the element. It had been previously shown (18) and was confirmed here that precipitation of ions during fixation is no safeguard to their persistence in the tissues after further processing. X-ray analysis can validate the visual observations and provide semiquantitative information on elemental concentration and distribution following different preparative treatments. The improvement brought about by the glutaraldehyde+urea embedding consists of its changing the fixed material less than do other embedding techniques. Washing out of substances is minimized because the polymerization progresses far more rapidly than dehydration. Analysis of minimally treated tissue showed that most physiological electrolytes are not detected satisfactorily even with glutaraldehyde +urea embedding under the instrumental conditions used here. However, chlorine and calcium can be roughly quantitated and are demonstrated visually with special methods of pretreatment. For X-ray microanalysis of chlorine in muscle, Epon cannot be used because it contains chlorine. Glutaraldehyde+urea embedding solves this problem. It also appears satisfactory for maintaining the distribution of histochemically precipitated ions better than Epon embedding in which postosmification and dehydration processes alter the picture. Except for the Osmium vapor, no staining was attempted in this study because the heavy metals used as stains may actually replace the ions of interest (18), or interfere with analysis by their own X-ray emission.

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Calcium detection and localization presented different problems. Calcium was detected with all methods of preparation in the nucleus, especially in electron-dense regions. Since little is known about calcium exchanges between the cytoplasm and the nucleus, this opens many possibilities for further investigations. Generally speaking, the detection of calcium in muscle by X-ray microanalysis is feasible. The EMMA-4 can detect 10-17 gram of a substance, and an elemental concentrations of somewhat less than 10-z in thin sections (100-200 nm). The concentration of calcium in blood or in whole hydrated heart tissue is 2.5 mM (equal to approximately 100 mg per liter or a mass fraction of about 10-4 on a weight-to-weight basis). In several intracellular loci, the calcium may be many times more concentrated, and thus should be detectable (7, 17). Osmium contracts muscle and carries large amounts of calcium into the cell (10). Although this is an unfortunate situation for absolute quantitation, it may be of advantage in any comparative study. The localization produced by pyroantimonate + osmium fixation further aids comparative, as well as morphological work. The origin of the calcium brought into the cell is not certain. Although the calcium might well come from without the cell, muscle left in calcium-free solutions for many hours still contracts on addition of the pyroantimonate and osmium solution and exhlbits the typical calcium antimonate precipitate (17). It seems possible that the osmium shifts calcium into the sarcomeres from cell-wall or membrane-bound intracellular loci. However, the nucleus may also be a source of sarcoplasmic calcium. The great scatter and variability in results of X-ray microanalysis of calcium in frog muscle and dog myocardium (16, 18) prompted us to find better methods of specimen preparation. The elimination of some artifacts by minimal dehydration with the method described in this paper appears to improve preparatory procedures for X-ray microanalysis and histochemistry. We wish to thank S. Schwartz and M. Scripps for technical assistance and B. Golek for editorial help. The funds for the instrumentation and the research were provided by the British Science Research Council, The Hebrew University-Hadassah Medical School and the European Molecular Biology Organization. REFERENCES 1. 2. 3. 4.

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5. HALL,T. A., ROCKERT,H. O. E. and SAUNDERS,R. L., X-Ray Microscopy in Clinical and Experimental Medicine. Thomas, Springfield, Illinois, 1972. 6. HALL, T. A., ANDERSON, H. C. and APPLETON, T. C., J. Microsc. 99, 177 (1973). 7. KLEIN, R. L., HORTON,C. R. and THURESON-KLEIN,A., Amer. J. Cardiol. 25, 300(1970). 8. KOMN1K,H., Protoplasma 55, 414 (1962). 9. KOMNIK,H. and BIERTHER,M., Histochemie 18, 337 (1962). 10. PAGE, E., J. Gen. Physiol. 51, 211s (1968). 11. PEASE, D. C. and PETERSON,R. G., J. Ultrastruct. Res. 41, 133 (1972). 12. PETERSON,R. G. and PEASE, D. C., J. Ultrastruct. Res. 41, 115 (1972). 13. TANDLER,C. J. and KIRSZENBAUM,A. L., J. Cell BioL 50, 830 (1971). 14. TANDLER,C. J., LIBANTI,C. M. and SANCHIS,C. A., 3". Cea Biol. 45, 355 (1970). 15. YAROM,R., BEN-ISHAY,D. and ZINDER, 0., J. Mol. Cell. CardioL 4, 559 (1972). 16. YAROM, R. and CHANDLER,J. A., J. Histochem. Cytochem. 22, 147 (1974). 17. YAROM, R. and MEIRI, U., J. Histochem. Cytochem. 21, 146 (1973). 18. YAROM,R., PETERS, P. D., ScRn, Ps, M. and ROGEL, S., Histochemie 38, 143 (1974).