a comparative study of three methods for the ... · j. cell sci. 9, 727-749 (i97i) 727 printed in...

J. Cell Sci. 9, 727-749 (i97i) 727Printed in Great Britain

A COMPARATIVE STUDY OF THREE METHODS

FOR THE ULTRASTRUCTURAL

DEMONSTRATION OF GLYCOGEN IN THIN

SECTIONS

M. V. VYE AND D. A. FISCHMANDepartment of Pathology, University of IllinoisCollege of Medicine, Chicago, Illinois 60612, U.S.A., andDepartments of Anatomy and Biology, University of Chicago,Chicago, Illinois 60637, U.S.A.

SUMMARY

In order to evaluate 3 staining methods for demonstration of glycogen in thin sections, 2tissues containing an abundance of this carbohydrate in /?-particle form were studied. Tissueswere aldehyde-fixed, postfixed in osmium tetroxide, embedded in Araldite and sectioned inthe usual manner without special precautions. The first method for staining thin sectionsemployed a sequential combination of periodic acid, thiosemicarbazide and osmium tetroxidevapour, while in the second procedure a silver protein solution was substituted for the osmiumtetroxide vapour. The third technique utilized periodic acid, sodium chlorite and uranylacetate, also in sequential combination. Each method yielded glycogen particles of greaterelectron density than were seen in sections stained by the usual uranyl acetate-lead citrateprocedure. Under high magnification, considerable method-dependent variation in theappearance of the glycogen granules was noted. Particulars substructure, only faintly visiblein routinely stained sections, was easily resolved with the periodic acid-thiosemicarbazide-silverprotein technique. Conversely, periodic acid-thiosemicarbazide-osmium tetroxide completelyobscured this substructure. With periodic acid-sodium chlorite—uranyl acetate, glycogenparticles appeared larger, more confluent, and of a less regular outline than with the othermethods. Sections were also stained by incubation in periodic acid prior to treatment withlead citrate. The alteration in appearance of the glycogen granules produced by this modificationwas so great that high-resolution analysis of particle size and substructure could not beundertaken. The usefulness of the procedures investigated here resides in their ability tostain glycogen in thin sections in an intense and selective manner.

INTRODUCTION

The ultrastructural demonstration of glycogen in thin sections of osmium-fixedtissue presented a considerable problem for early workers in electron microscopy.This difficulty resulted from the paucity of chemical groups in glycogen capable ofbinding or reducing osmium tetroxide (Revel, 1964). Thus, even if glycogen particleswere structurally preserved by osmium fixation, their electron density was so lowthat effective visualization was not possible (Fawcett, 1955; Nilsson, 1962). Doublefixation with aldehydes and OsO4 did not obviate this problem, for although aldehydesimproved structural preservation, the glycogen particles were still of insufficientelectron density to be adequately resolved.

728 M. V. Vye and D. A. Fischman

Successful efforts to find an electron-dense stain for glycogen began with thereport by Swift & Rasch (1958) of the staining of glycogen particles in osmium-fixedtissues by phosphotungstic and phosphomolybdic acid. Watson (1958 a) confirmedthis finding with phosphomolybdic acid, but in a later report (19586) showed thatlead hydroxide was a superior stain for glycogen. Subsequently, the classic study ofRevel, Napolitano & Fawcett (i960) conclusively demonstrated that glycogen particleswere stained intensely by lead hydroxide after either osmium tetroxide or potassiumpermanganate fixation. Lead citrate was shown to have similar properties (Reynolds,

Though quite effective for demonstrating glycogen, lead solutions were non-specific stains which enhanced the contrast of many cellular components (Revel, 1964)and did not reveal or reflect any of the specific chemical properties of glycogen.Consequently, attempts were made to develop a selective staining reaction forpolysaccharides. A number of investigators (Suzuki & Sekiyama, 1961; Movat, 1961;Steiner & Carruthers, 1961; Marinozzi, 1961) adapted the periodic acid-methenaminesilver method of Gomori (1952) for staining of polysaccharides in plastic-embeddedthin sections, and Movat (1961) was successful in demonstrating glycogen with it.This finding, though disputed by Rambourg & Leblond (1967), has been confirmedindependently by Marinozzi (1963) and Thiery (1967). However, the technique wasalso non-specific, for the silver reaction product was deposited on many intracellularstructures, particularly membranes (Marinozzi, 1961). Furthermore, the coarsegranularity of the precipitate precluded high-resolution analysis of the glycogenparticles (Movat, 1961; Marinozzi, 1961; Thie"ry, 1967).

Subsequently, Hanker et al. (1964) introduced the osmiophilic reagent thiosemi-carbazide (TSC) for the ultrastructural demonstration of macromolecules in thinsections. This reagent binds to 1,2-dialdehyde groups generated by the periodic acidoxidation of carbohydrates and subsequently reduces osmium tetroxide to electron-dense osmium black. The specificity of this reaction is similar to the PAS stain asused in light microscopy (Seligman et al. 1965; Hanker et al. 1965). Intense, selectivestaining of glycogen particles in thin sections of fixed tissue was produced by thisprocedure (Hanker et al. 1965).

The ultrastructural methods for staining glycogen were reviewed by Thie>y (1967),who introduced a modification of the technique of Hanker et al. (1964) in whicha solution of silver protein replaced OsO4 vapour. This reagent, by eliminating theneed for osmium tetroxide vapour, greatly increased the convenience of the method.In addition, the staining produced by this modification was particularly delicate andrevealed a definite substructure in the glycogen particles.

Recently, in a study of glycogen-rich tissues, we have employed 3 methods forstaining glycogen particles; 2 involved the use of TSC while the third utilized sodiumchlorite and uranyl acetate. Though each procedure clearly demonstrated the glycogengranules, many differences were noted in their ultrastructural appearance dependingupon the method used. These findings are the subject of this report.

TJltrastructural demonstration of glycogen 729

MATERIAL AND METHODS

Preparation of tissue for electron microscopy

Two glycogen-rich tissues were investigated. The first, human skeletal muscle from threepatients with Type V glycogenosis, commonly called McArdle's disease (McArdle, 1951),was obtained by open biopsy under local anaesthesia and maintained at rest length in a muscletension clamp (Price, Howes, Blumberg & Pearson, 1965). The biopsies were immediatelyfixed for 2 h in 2-5 % glutaraldehyde or 4% paraformaldehyde in Millonig's buffer (Millonig,1961) at pH 74. Following initial fixation, blocks measuring 1 mm3 were dissected from thesurfaces of the specimens and fixed for an additional 12 h. The blocks were stored in bufferfor a period not exceeding 24 h prior to embedding. The second tissue, chick glycogen body,was obtained from the lumbar enlargement of the spinal cord of 20-day White Leghorn chickembryos. After being cut into i-mms blocks, they were fixed and stored in the same manneras the skeletal muscle.

Following primary fixation, both tissues were postfixed in 1 % osmium tetroxide in Millonig'sbuffer for 1 h, after which they were dehydrated in graded ethanols and embedded in Araldite.One-micron thick sections for light-microscopic survey were cut with glass knives and stainedwith toluidine blue-O. Thin sections were cut with diamond knives and mounted on uncoatedcopper or gold grids.

Staining methods for glycogen

The 3 methods used for the demonstration of glycogen required periodic acid oxidation.For these procedures sections were mounted on inert gold grids. All steps of the staining,except rinsing in H.O, were performed by floating the grids on a drop of reagent solution.(Reagents were purchased from the following suppliers: thiosemicarbazide, Eastman OrganicChemicals; silver protein (mild), Merck and Co.; lead citrate, K and K Laboratories, Inc.;uranyl acetate, Fisher Scientific Co.; sodium chlorite, Matheson, Coleman and Bell. Periodicacid and osmium tetroxide were obtained from several sources.)

Periodic acid-thiosemicarbazide-osmium tetroxide (PA-TSC-OT). The method describedhere is essentially similar to the technique published by Hanker et al. (1964). Thin sectionsmounted on gold grids were incubated in 1 % periodic acid for 30 min, following which theywere rinsed for 1 min in a jet of distilled water. The sections were then treated for 60 minwith a 1 % solution of thiosemicarbazide dissolved in 25 % acetic acid. Following a secondrinse in distilled water, the sections were reacted with osmium tetroxide vapour by mountingthe grids on coverslips which were placed in small Coplin jars containing OsO4 crystals. Thecontainers were sealed with stopcock grease and incubated for 3 h in a waterbath at 60 °C tovolatilize the osmium.

Periodic acid-thiosemicarbazide—silver protein (PA-TSC-SP). This method, a modificationof the previous technique, was reported by Thidry (1967). The procedure given here differsfrom his description in that TSC was dissolved in 25% acetic acid rather than 10%, thatsections were rinsed with distilled water rather than acetic acid, and that sections were mountedon grids before staining instead of after it. Using this method, sections were reacted withperiodic acid, rinsed in distilled water, incubated in thiosemicarbazide and rinsed again ina manner identical to the PA-TSC-OT technique. At this point the sections were treatedwith 1 % aqueous solution of silver protein for 30 min followed by a final i-min rinse witha jet of distilled water. The silver protein reagent was prepared fresh and used immediatelyin the dark.

Periodic acid-sodium chlorite-uranyl acetate (PA-SC-UA). This technique was developedby M. J. Kamovsky & J. P. Revel (personal communication) but has not been published.Sections were reacted with 1 % periodic acid for 30 min and rinsed with distilled water as inthe other methods. Subsequently, they were incubated for 15 min in a 2-5% solution ofsodium chlorite dissolved in 10% acetic acid. The sections were again rinsed, followingwhich they were treated for 10 min with a saturated aqueous solution of uranyl acetate. A finalrinse in a jet of distilled water completed the procedure. The sodium chlorite reagent mustalso be prepared fresh and used in the dark.

730 M. V. Vye and D. A. FischmanOther grids were stained by the conventional method employing lead citrate in sequential

combination with saturated uranyl acetate in 50% alcohol (UA-LC). Lead citrate stainingwas also employed alone following oxidation with 2-5 % periodic acid for 30 min, as recom-mended by Marinozzi (1963) and modified by Perry (1967).

Two sets of control grids employing incomplete staining reactions were prepared. In thefirst, water was substituted for periodic acid, while in the second, water or acetic acid wassubstituted for thiosemicarbazide (first 2 methods) or sodium chlorite (last method).

OBSERVATIONS

McArdle's disease (McArdle, 1951) is an uncommon type of glycogenosis whichaffects skeletal muscle and is due to a genetically determined deficiency of the glyco-lytic enzyme phosphorylase (Schmid & Mahler, 1959; Pearson, Rimer & Mommaerts,1959). The ultrastructure of skeletal muscle in this condition was first described bySchotland, Spiro, Rowland & Carmel (1965). Several subsequent reports (Delwaideet al. 1967; Boudouresques et al. 1967; Brownell, Trevor-Hughes, Goldby & Woods,1969) have confirmed their findings, and the appearance of the tissues studied heredoes not differ significantly from that described in these reports. The muscle fibresare distorted by large subsarcolemmal and intermyofibrillar aggregates of glycogen(Fig. 1). These intermyofibrillar pools occur mainly in the I-band, but the numberof particles located within the myofibrils is also increased. There is no constantassociation of the particles with intracellular membranes, nor are the glycogenaggregates membrane-bound. Individual granules resemble the /?-particles describedby Drochmans (1962). The biochemical structure of the glycogen molecule in thiscondition has been reported to be normal (Mommaerts et al. 1959).

The ultramicroscopic appearance of the embryonic chick glycogen body has beendescribed by Revel et al. (i960). The cytoplasm of the cells consists of a large poolof glycogen particles. Nuclei are peripherally placed and a few organelles are locatedin a perinuclear position (Fig. 2).

In the 3 methods used for the selective staining of glycogen, sections were oxidizedwith periodic acid but not treated with lead solutions. Such sections, when comparedwith tissue stained by the usual uranyl acetate-lead citrate (UA-LC) procedure,exhibited glycogen particles which appeared to be of greater electron density thanwith the routine method (Figs. 3-8). At low magnification (less than x 15000) resultswith the 3 stains appeared quite similar. Since periodic acid oxidation removesmuch of the reduced osmium from the tissues (Merriam, 1958; Seligman, 1966),glycogen particles were revealed as densely stained globular bodies against a palebackground.

Though each method stained glycogen intensely, differences were noted in thesize and configuration of the granules. In Table 1 the size distribution of glycogenparticles from both tissues is presented for each method as well as for routine UA-LCstaining. Even though the range of particle size was quite great, certain pertinentobservations can be made. In all cases, the average particle diameter was greaterin the glycogen body than in muscle. With each method, the difference between the2 tissues was statistically significant (P < o-oi). A further observation was thatparticle diameter in both the glycogen body and skeletal muscle, when measured

Ultrastructural demonstration of glycogen 731

after PA-SC-UA staining, was significantly larger than with the other techniques(P < o-oi).

In addition to these quantitative variations, there were qualitative differences inthe appearance of the glycogen granules. In sections conventionally stained withuranyl acetate and lead citrate, glycogen appeared as discrete or partially interconnectedspheroidal particles which demonstrated a faint but constant substructure (Figs. 9, 10).With both PA-TSC-OT and PA-TSC-SP, the general outline of the particles wasmaintained, but no substructure could be seen with certainty in the PA-TSC-OTmaterial (Figs. 11, 12). Glycogen stained with PA-TSC-SP, however, revealed adefinite substructure within the granules which was similar to that seen in UA-LCstained particles, but more easily resolved (Figs. 13, 14). Each particle appeared tobe composed of globular subunits measuring about 3 nm in diameter (inset, Fig. 14).With the PA-SC-UA method glycogen granules appeared as a series of interconnectedunits which were quite variable in size and shape (Figs. 15, 16), and usually exhibiteda light centre which gave them a doughnut-like appearance (inset, Fig. 16). Theglobular subunits were not well defined by this technique.

Table 1. Diameter of glycogen particles with various staining methods

Staining method

UA-LC

PA-TSC-OT

PA-TSC-SP

PA-SC-UA

Mean diameter (± 2 S.D.)and range in

human skeletal muscle, nm

26-414-7(18-0-37-5)

26-414-2(20-5-38-5)

29-1 ±3-3(22-0-36-0)

35'8±6-2(20-5-53-5)

Mean diameter ( ± 2 S.D.)and range in

chick glycogen body, nm

37-1 ±7-2(25-0-54-0)35-1 ±6-o

(27-5-49-0)

38-1 ±6-5(28-5-53-0)

47-8 ±9-i(29-5-65-0)

When grids were stained with lead citrate alone following oxidation with 2-5%periodic acid, as recommended by Perry (1967), the structure of glycogen wasaltered considerably (Figs. 17, 18). Margins of the /^-particles were obscured, andin their place a continuum of irregularly sized granules measuring between 3 and24 nm in diameter was observed. The smallest particles resembled subunits seenwith UA-LC and PA-TSC-SP staining; however, it was difficult to relate them toa larger particulate structure. Because of this highly variable appearance, size analysisof the y?-particles could not be performed.

In control specimens employing an incomplete staining protocol, glycogen didnot appear electron-dense. The particles presented instead as electron-lucent bodiesagainst a darker background (Figs. 19, 20). Thus, the pictures produced by theincomplete staining procedures were virtually the negative image of the completereactions.

47 C E L 9


DISCUSSION

Though lead solutions produce satisfactory delineation of glycogen particles inmost situations, the non-specific nature of this staining method has long beenrecognized (Revel, 1964). In addition, lead staining of glycogen is often unpredictable,considerable variation in density being noted with different preparations of reagentand even with the same preparation at different times. In view of these difficulties,3 methods for staining glycogen which do not employ lead have been investigated.Both the PA-TSC-OT and PA-TSC-SP techniques are based on a principle similarto the light-microscopic PAS stain (Hanker et al. 1965; Seligman et al. 1965). Thus,they are capable of staining substances with dialdehyde linkages or chemical groupswhich can be converted to dialdehydes by periodic acid oxidation. The PA-TSC-OTmethod has been used to study the extraction of glycogen by diastase digestion fromEpon-embedded liver tissue (Rosa & Johnson, 1967), the distribution of glycogenin McArdle's disease (Vye, 1968), extraction of glycogen by en bloc staining withuranyl acetate (Vye & Fischman, 1970), and the structure of Armanni-Epsteinlesions in renal tubular cells of animals with experimental diabetes (Vye, Friederici& Vargas, submitted for publication). The PA-TSC-SP technique has been employedin the investigation of mucopolysaccharide synthesis by the Golgi apparatus ofintestinal epithelial cells (Thi^ry, 1969), and to localize glycogen in spermatozoa(Anderson & Personne, 1970). The histochemical basis of the PA-SC-UA techniquehas not been clearly elucidated, nor is it known if its reaction mechanism is similarto the 2 methods discussed above.

When the diameters of glycogen particles in the chick glycogen body and musclefrom patients with McArdle's disease were compared, it was found that with all4 staining methods the granules were significantly larger in the glycogen body.Revel et al. (i960) reported an average diameter of glycogen particles in permanganate-fixed, lead-hydroxide-stained chick glycogen body to be about 30 nm. However, henoted considerable variability of particle size, with a range from 17 to 100 nm indiameter. In two cases of McArdle's disease, Schotland et al. (1965) reported thatglycogen particles ranged from 15 to 50 nm in diameter, but did not report a meanvalue. Biava (1963), in a study of many human tissues including skeletal muscle,stated that the mean diameter of monoparticulate glycogen varied from 22 to 30 nmfollowing osmium tetroxide fixation and lead hydroxide staining. A specific figurefor skeletal muscle, however, was not given. Wanson & Drochmans (1968a), on theother hand, reported a mean diameter of glycogen particles in glutaraldehyde-fixed,lead-hydroxide-stained rabbit muscle to be 27-3 + 3 nm. Therefore, the particle sizenoted here for lead-stained skeletal muscle is in good agreement with previousinvestigations. With chick glycogen body the mean particle diameter of 37-1 nmreported in this study is somewhat larger than the 30 nm given by Revel et al. (i960).However, in view of the large range of particle size in both studies, such a discrepancyis not surprising. Because the difference in particle size between glycogen body andskeletal muscle has been noted with all staining methods, it is probable that thesefigures reflect a real difference in size of the glycogen granules between the 2 tissues.


Perhaps the most interesting question raised by these results relates to themethod-dependent appearance of the glycogen particles in sectioned tissue. Theclearest elucidation of glycogen /7-particle structure has been obtained from thestudy of negatively stained preparations of isolated glycogen from both rat liver(Drochmans, 1962) and rabbit skeletal muscle (Wanson & Drochmans, 1968a), inwhich the granules were found to be composed of small, regular subunits about3 nm in diameter. Limitations imposed by thin sectioning make analysis of particlestructure in fixed tissue considerably more difficult. Section thickness is hard tocontrol precisely and granularity of the image at high magnification may be confusedwith particle substructure. Despite these problems, a substructure similar to thatseen in negatively stained particles was present in lead-stained material. This is inaccord with the previous findings of Revel et al. (i960) and Biava (1963). Stainingwith PA-TSC-SP improved the resolution of the subunits, as previously noted byboth Thiery (1967) and Anderson & Personne (1970). In view of this fact, it iscurious that the PA-TSC-OT method, which is based on the same chemical principle,not only fails to amplify the substructure, but actually obscures it.

With PA-SC-UA, the particles were significantly larger and had a somewhatdifferent appearance; only a faint substructure was visible. These results raise thequestion of what is actually being stained by the various methods. In rat liver,isolated glycogen particles have been shown to have enzymically active proteinbound to them (Leloir & Goldemberg, i960; Luck, 1961). Wanson & Drochmans(1968 a) were unable to reduce the protein content of isolated rabbit muscle glycogenparticles below 3 % without destroying the structural integrity of the particles, andthese protein-containing particles were demonstrated to possess phosphorylase activity(Wanson & Drochmans, 19686). Thus, it seems likely that glycogen in the cellexists as a protein—polysaccharide complex in which some of the enzymes of glycolysisare present. The PA-TSC-OT and PA-TSC-SP procedures are based on thereaction of dialdehydes produced by periodic acid oxidation. Consequently, withthese methods the polysaccharide, which contains many diol groups, should be themajor component that is stained. On the other hand, uranyl acetate, at least whenused alone, is a relatively non-specific stain capable of reacting with proteins andnucleic acids (Watson, 1958 a). It is possible, therefore, that the unusual configurationof glycogen particles seen with PA-SC-UA staining may result from staining ofprotein as well as the polysaccharide component of the glycogen particle.

Further evidence for the method-dependent appearance of the glycogen particleswas provided by the curious morphological changes that were wrought when leadstaining was preceded by periodic acid oxidation. The chemical basis of thesealterations is obscure.

The principal advantage of the techniques investigated here is that each providesa selective method for the demonstration of glycogen in thin sections. Furthermore,unlike the methods described by de Bruyn (1968) and Bradbury & Stoward (1967,1968), special preparation of the tissues during fixation or prior to embedding arenot required. The PA-TSC-SP method produces an ultrastructural configurationof the glycogen particles which is in accord with data from negative staining and

47-2


with the appearance of the granules in lead-stained sections. The PA-TSC-OTmethod has been quite useful in differentiating glycogen from ribosomes in sectionsof liver which have been double-fixed with glutaraldehyde and osmium tetroxide(M. V. Vye, unpublished observations). It is not possible, however, to state withcertainty which stain, if any, demonstrates glycogen as it truly exists in situ. Themajor disadvantages of the methods relate to the time required for preparation ofreagents and staining, as well as the necessity of using uncoated inert grids. Thelack of staining of other tissue components may be a disadvantage in some situations,although this is not the case when the selective effect of a 'special stain' is sought.

Because of their ability to stain polysaccharides selectively in tissues fixed andembedded by standard techniques, these cytochemical methods should be of valuein many areas of ultrastructural investigation.

The authors wish to express their appreciation, to Misses Rita Yambot, Irena Kairys andMrs Lucy Vedegys for their excellent technical assistance. This work was supported byGrant No. 315 from the General Research Support Grant of the University of Illinois (toM.V.V.) and No. GB-7591 from the National Science Foundation and No. N-69-32 fromthe Chicago and Illinois Heart Association (to D.A.F.).

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WATSON, M. L. (19586). Staining of tissue sections for electron microscopy with heavy metals.II. Application of solutions containing lead and barium. J. biophys. biochem. Cytol. 4,727-730.

{Received 22 March 1971)

Fig. 1. Skeletal muscle from a patient with Type V glycogenosis stained with uranylacetate and lead citrate. Large aggregates of particulate glycogen are located beneaththe sarcolemma and between the myofibrils. The number of particles between myo-filaments within the myofibrils is also increased. A, A-band; /, I-band; Z, Z-line;g, glycogen; m, mitochondrion, x 15000.Fig. 2. Glycogen body from 20-day chick embryo stained with uranyl acetate andlead citrate. The cytoplasm of the cells consists almost entirely of a large pool ofglycogen particles, g, glycogen; m, mitochondrion; n, nucleus, x 15000.


Fig. 3. Skeletal muscle as in Fig. 1 stained by the periodic acid-thiosemicarbazide—osmium tetroxide (PA-TSC-OT) method. The electron-dense glycogen particlesstand out in sharp relief against the paler structures of the muscle fibre. Though muchreduced in electron density, the structure of the mitochondria and myofibrils is stilldiscernible. A, A-band; /, I-band; Z, Z-line; g, glycogen; m, mitochondrion,x 15000.

Fig. 4. Glycogen body as in Fig. 2 stained by the PA-TSC-OT procedure. Electron-dense glycogen granules dominate the ultrastructural appearance. The shadow of anucleus is faintly visible, g, glycogen; n, nucleus, x 15000.


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Fig. 5. Skeletal muscle as in Fig. 1 stained by the periodic acid-thiosemicarbazide-silver protein (PA-TSC-SP) method. The tendency of glycogen to aggregate in theI-band is apparent in this section. A, A-band; /, I-band; Z, Z-line; g, glycogen;m, mitochondrion, x 15000.Fig. 6. Glycogen body as in Fig. 2 stained by the PA-TSC-SP technique. Thesection is composed almost entirely of densely stained glycogen particles, g, glycogen.x 15000.

Fig. 7. Skeletal muscle as in Fig. 1 stained by the periodic acid—sodium chlorite—uranyl acetate (PA-SC-UA) procedure. The similarity to Figs. 3 and 5 is apparent.A, A-band; /, I-band; Z, Z-line; g, glycogen; m, mitochondrion, x 15000.Fig. 8. Glycogen body as in Fig. 2 stained by the PA-SC-UA method. The densityof the glycogen granules is similar to Figs. 4 and 6. g, glycogen; m, mitochondrion,x 15000.


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Fig. 9. Skeletal muscle stained with uranyl acetate and lead citrate. The glycogenparticles appear as a series of interconnected granules in which a faint substructurecan be discerned, mf, myofibril. x 100 000.Fig. 10. Glycogen body stained with uranyl acetate and lead citrate. The glycogenparticles are also seen as a series of interconnected granules displaying a faint sub-structure. At higher magnification (inset) the substructure is well resolved and showsthe glycogen granules to be composed of subunits measuring about 3 nm in diameter.When compared with Fig. 9, the larger size of the particles in the glycogen body isapparent, x 100000; inset, x 250000.

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Fig. i i . Skeletal muscle stained by the PA-TSC-OT technique. Though glycogenparticles are well stained, no substructure is noted, wf, myofibril. x iooooo.Fig. 12. Glycogen body stained by the PA-TSC-OT procedure. Electron-denseinterconnected glycogen particles are clearly delineated. Even at high magnification(inset) a well-defined substructure cannot be resolved. The larger size of the particlesin the glycogen body as compared to skeletal muscle (Fig. 11) is apparent, x iooooo;inset, x 250000.

Fig. 13. Skeletal muscle stained by the PA-TSC-SP method. The glycogen particleshave an easily resolvable substructure which is composed of globular units measuringabout 3 nm in diameter, x iooooo.Fig. 14. Glycogen body stained by the PA-TSC-SP technique. The glycogenparticles also have a clearly resolved substructure composed of small globularfragments which measure about 3 nm in diameter; at high magnification (inset) thissubunit is quite similar to that seen in the uranyl acetate-lead citrate stained tissue(Fig. 10). The larger size of the glycogen granules in the glycogen body is also apparent,x iooooo; inset, x 250000.

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Fig. 15. Skeletal muscle stained by the PA-SC-UA method. The glycogen particlesassume a less regular, more interconnected appearance. Substructure within thegranules is not clearly visualized, m, mitochondrion; mf, myofibril. x 100000.Fig. 16. Glycogen body stained by the PA-SC-UA procedure. The glycogen particleshave an irregular outline and are more highly interconnected. Many granules havea lucent centre, imparting a doughnut-like appearance. Substructure is not wellresolved. Because of the irregular shape, differences in particle size between muscleand glycogen body are less apparent with this procedure, x 100000; inset, x 250000.Fig. 17. Skeletal muscle stained with lead citrate preceded by incubation in periodicacid. Though glycogen stains intensely, the particulate structure has been greatlyaltered. In such sections glycogen appears to be composed of granules which varyin size from 3-0 to 24 nm. The smallest of these resemble the subunits seen insections stained with uranyl acetate and lead citrate (Fig. 9) or PA-TSC-SP (Fig. 13),though it is difficult to relate these units to any larger structural component, mf,myofibril. x 100000.Fig. 18. Glycogen body stained with lead citrate preceded by incubation in periodicacid. The uJtrastructural alterations of the glycogen particles in this tissue resemblethose seen in muscle treated in a similar manner (Fig. 17). x 100 000.

Ultrastructural demonstration of glycogen

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Fig. 19. Glycogen body stained by the PA-TSC-OT technique but with H2Osubstituted for PA. The glycogen particles appear as electron-lucent globules againsta dark background, g, glycogen. x 15000.Fig. 20. Glycogen body stained by the PA-TSC-OT procedure but with H8Osubstituted for TSC. Glycogen again appears as electron-lucent particles againsta dark background. Other staining methods using an incomplete reaction protocolyielded glycogen particles of similar electron density, g, glycogen. x 15000.

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a comparative study of three methods for the ... · j. cell sci. 9, 727-749 (i97i) 727 printed in...

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