448

A Comparative Study of and Histochemical Reactions of the Components of Golgi Complex of the Fibroblasts in vitro and the Ascites Tumor Cells

Shunzo Takagi, Jin-ichi Kitada, Hideo Masuda and Masayuki Tagawa Departmentof Biology,University of OsakaPrefecture, Sakai,Japan ReceivedJune 27, 1961

It was previously shown with phase optics that the Golgi apparatus in living cells cultured in vitro, such as fibroblasts, corneal epithelial cells, myoblasts and white cells of the domestic fowl, consists of canalicules, which are usually seen as vacuoles in optical section, and of delicate filaments in a juxtanuclear area distinguishable from the rest of the cytoplasm (Takagi and Masuda 1956, Takagi 1958). Similar canalicules and filaments were also found to constitute the Golgi apparatus of frog lymphocytes (Takagi and Tagawa 1957). The walls of canalicules and the filaments are readily black ened by osmium tetroxide and the whole apparatus appears like a congeries of black strands. They are also stained vitally with Janus green and , and vitally and metachromatically with , trimethylthionin and toluidine blue. The vital staining indicates that a canalicule is a dilated filament or often a dilated part of a filament. A number of electron microscopists have revealed that the Golgi complex consists of vacuoles, smooth membranes and small vesicles or granules, and the vacuoles are a dilatation of a double membrane, being characteristic of an actively secreting or absorbing cell (Haguenau and Bernhard 1955, Gatenby and Lufty 1956, Sjostrand 1956, Dalton and Felix 1956, 1957, Lacy 1957). The canalicules and the filaments observable in the living cells under the phase microscope may in all probability correspond to the vacuoles and the smooth membranes respectively in the electron micrographs, in which the latter often appear in the form of flattened vesicles and tubules. The other component of the Golgi complex, the small vesicles, being too small, are not to be seen under the phase microscope. As will be described later, the canalicular and the filamentous elements of the Golgi apparatus in cells vitally stained or stained after fixation both appear under the ordinary microscope as strands or filaments with various diameters. Hence, in the following lines, they will be referred to for convenience' sake as Golgi filaments. Our present knowledge on the staining and histochemical properties of the Golgi apparatus is based for the most part on studies of the structure identifiable as such by observing and comparing some standard Golgi prepara- 1961 Staining and Histochemical Reactions of Golgi Complex 449 tions with it. Elements of the Golgi complex directly observed in the living cell do not seem to have been the object of extensive study . Our present investigation aims at the study of whether the Golgi filaments, directly ob servable in the cultured fibroblasts and the mouse ascites tumor cells, are vitally stained, and after fixation are stained and whether they indicate any definite histochemical properties. The conspicuous result among many ob tained is that the Golgi filaments behaved themselves tinctorially very much like the mitochondria, although the former were distinct from the latter in some other features. The present investigation was made upon the basis of our observations of the Golgi apparatus in the two types of cells in living unstained condition, which will also be described.

Materials and methods

Fibroblasts cultivated from the heart of chick embryos and ascites tumor cells of the EM strain of the mouse were used. The former were derived from 8- to 9-day embryos and cultured in the medium of homologous em bryonal extract and plasma with the hanging drop method. The latter were supplied by the Zoological Institute of Hokkaido University. We observed in the previous study (Takagi and Masuda 1956) the Golgi apparatus of the fibroblasts in the living condition, after the addition of one per cent solution of osmium tetroxide and also in the Kolatchev preparation, and the organelle of the cells vitally stained with Janus green, neutral red and the other dyes. These observations were not, therefore, repeated in the present study. The ascites cells were observed with the use of a NDM phase objective (Tiyoda) in the ascites fluid mixed with an equal volume of a standard solu tion of powdered human serum. The Golgi filaments could be clearly dis tinguished from the mitochondria by this method. For the Kolatchev preparation, a drop of the ascites fluid was smeared on a coverglass, fixed in Champy and impregnated in one per cent osmium tetroxide. The direct action of osmium tetroxide on the Golgi filmaments of the ascites cells was examined under the phase microscope with the addition of a drop of one per cent solution to a drop of ascites fluid. Fibroblasts cultured on a coverglass and ascites cells smeared on a coverglass were fixed in Champy's fluid and stained with Altmann's acid fuchsin and picric acid with or without postchromation. In the vital staining of the tumor cells with Janus green, a drop of a 1:5,000 solution of the dye in Ringer was added to a nearly equal drop of the ascites fluid. The other vital dyes were applied also in a similar manner. The cells of two kinds on a coverglass were stained with Heidenhain's iron hematoxylin after being fixed in Champy's fluid and chromated in 2 per cent potassium bichromate. They were also stained with resorcin-fuchsin 450 S. Takagi, J. Kitada, H. Masuda and M. Tagawa Cytologia 26 and counterstained with carmalum after fixation in 15 per cent formalin. The same materials were fixed in calcium-formol and were, after stain ing of the nucleus with carmalum or Delafield's hematoxylin, stained with sudan black. Fixed in the same manner, they were treated with trypsin and stained with Nile blue according to Tarao's procedure (1939) for the detec tion of masked lipid. This method was only applicable to the tumor cells, because the fibroblasts easily detached themselves from the coverglass during the treatment with trypsin. Baker's acid hematein test was employed for investigating phospholipid content of those two kinds of cells. Periodic acid-Schiff reaction after McManus was applied to the materials fixed in Gendre's fluid and digested with amylase. Alkaline phosphatase activity was tested according to Gomori's method.

Results

In phase microscopic observations of the fibroblasts in vitro, it was re peatedly seen that a clump of vacuoles and filaments, embedded in a ground substance which usually appears darker than the general cytoplasm, is always

Fig. 1. Semi-diagram of the fibroblast (a) and the ascites tumor cell (b), showing the authors' interpretation of the structure of Golgi complex (in the figure, above the nucleus). Mitochondria and fat droplets are also shown. situated on one side of the nucleus. The vacuoles are 10-20 in number and usually 0.3-0.5 micron in diameter, and are in extreme cases as large as 1.0 micron. It was confirmed that the vacuoles represent optical sections of canalicules. The filaments run in various directions, but in elongated cells they are orientated almost parallel to the longer axis of the nucleus (Figs. la, 2). That the vacuolar and filamentous structures represent the elements of the Golgi complex of the electron microscopists is evident from the results of our previous study (Takagi and Masuda 1956) and from the facts to be 1956 Staining and Histochemical Reactions of Golgi Complex 451 described in the following pages. The ascites tumor cell shows under the phase microscope an area, lighter than its surroundings, of the cytoplasm on one side of the nucleus and usually at the center of the cell. In the area, filaments, extremely fine and gray, are seen running in various directions. They are thought to represent Golgi elements of the cell. There are no vacuoles in this area. The mito chondria in the form of short threads are orientated at random and are distributed in the peripheral cytoplasm, where a variable number of lipid droplets are found (Fig. 1b). In Kolatchev preparations of the fibroblasts, the Golgi apparatus consists of a clump of black threads of variable diameter, which are richly entangled, but do not form a real network (Takagi and Masuda 1956). In the ascites cells, it is likewise composed of thick black threads without anastomosis (Fig. 3). In some cells, the ground substance of the Golgi area is darkened with osmium and fine filaments are gray in it. In this case, the Golgi filaments are only slightly osmicated and their blurred contour is preserved. The mitochondria are usually not blackened in the same degree as the Golgi filaments. The lipid droplets are stained black. That the elements of the Golgi ap paratus are usually blackened with osmium Fig. 2. Living fibroblasts observed more easily than the mitochondria was with phase contrast. A cell to the left has a Golgi area on the left side shown only by the addition of one per cent of the nucleus, which is darker than

solution of osmium tetroxide at room tem the surrounding general cytoplasm perature. Under the phase microscope, the and exhibits several Golgi vacuoles Golgi canalicules of the fibroblasts become within its confines. The other cell to the right has a similar Golgi area dark soon and appear like curved black in the upper right corner of the

rods, which show a light unstained center nucleus. Mitochondria are shown in optical sections. It shows evidently that as threads, ranging from gray to the walls of the Golgi canalicules or vacuoles black. The cells contain also nu merous fat droplets. •~750 have reduced osmium tetroxide. The fila mentous Golgi elements also turn gray (Takagi and Masuda 1956). Also in the ascites tumor cells, the addition of osmium tetroxide blackens Golgi filaments, but does not blacken mitochondria. This blackening is seen with difficulty under the ordinary microscope, but under the phase microscope a congeries of the Golgi filaments, which are black and entangled with each other, was found to present close similarity to the Golgi apparatus demon strated with the Kolatchev method (Fig. 4). It must be noted that each Golgi filament is far slender than a which appears like a black thread, and has a small dilatation at one or both ends and, more rarely, in 452 S. Takagi, J. Kitada, H. Masuda and M. Tagawa Cytologia 26

Figs. 3-8. 3, a Kolatchev treated tumor cell. The Golgi complex, composed of intensely blackened strands, is seen to the left of the nucleus at the center of the cell. Mitochondria, also blackened, are distributed mainly in the peripheral cytoplasm. Under the ordinary microscope. •~1,800. 4, a tumor cell with a congeries of blackened Golgi filaments in the concavity of the nucleus. A few mitochondria are shown in the peripheral cytoplasm

Reaction to 1 per cent solution of osmium tetroxide, observed under the phase microscope. 1961 Staining and Histochemical Reactions of Golgi Complex 453

the intermediate portion. The Golgi ground substance, which appears light with the phase microscope in the living cell, grows dark after the action of osmium tetroxide.

In the cells of two k minds, which were fixed in Champy, but were not

postchromed, Altmann's acid fuchsin stains red the Golgi ground substance, in which the more heavily stained Golgi filaments stand out clearly. The

staining of the Golgi filaments and the ground substance is more marked in the ascites cells than in the fibroblasts. The mitochondria, of course, are

also stained red. But the Golgi filaments are finer than the mitochondria,

but are not so smooth in outline as the latter (Fig. 5). When the materials

are postchromed, the mitochondria are more intensely stained than otherwise

and the Golgi filaments and ground substance, though stained, less attract

our attention.

In the fibroblasts, Janus green stains the mitochondria alone in a con

centration of 1:20,000, but stains the Golgi filaments as well in a concentra

tion of 1:10,000. It was found that in some cells a few mitochondria

thrust themselves in the Golgi area (Takagi and Masuda 1956). When the

ascites cells are stained with Janus green, the mitochondria which are mostly

rod-like are stained and, besides, a clump of filamentous structures with

blurred contour is stained in the concavity of the nucleus. The filaments

run mostly, though not unexceptionally, parallel to the longer axis of the

nucleus. There is hardly any doubt that they are elements of the Golgi

apparatus.

In parallel with the observation on the fibroblasts (Takagi and Masuda

1956), it was confirmed that neutral red, methylene blue, trimethylthionin

and toluidine blue can vitally stain the Golgi area and sometimes the Golgi

filaments in it in the ascites tumor cells, the last three dyes metachromati

cally staining them in color ranging from purple to red.

Iron hematoxylin stains the Golgi filaments slightly, if ever, in the

fibroblasts and not at all in the tumor cells, while it stains the mitochondria

•~1,600. 5, tumor cells stained with Altmann's acid fuchsin. A dark mass at the center of each cell represents the Golgi complex and consists of stained filaments with blurred contour. The nucleus, lightly stained, is situated in the upper left part of each cell. Some mitochondria are shown around the Golgi area and in the peripheral cytoplasm. Fixed in Champy's fluid and stained with acid fuchsin and picric acid. •~1,600. 6, tumor cells stained with sudan black. Fat droplets and mitochondria are intensely black. The nucleus appears lighter than the rest of the cell. Golgi filaments are faintly stained. Fixed in calcium-formol. Nuclear stain with carmalum. •~1,600. 7, fibroblasts stained with the PAS technique. In the left cell, a congeries of Golgi filaments, stained red faintly, is shown to the right of the nucleus, facing its indentation. Mitochondria are strongly colored. Fixed in Gendre's fluid; not digested with amylase; counterstained with Delafield's hema toxylin. •~900. 8, a tumor cell stained with the PAS technique. Mitochondria and seem ingly endoplasmic sacs are intensely colored. The Golgi area at the center and above the light-appearing nucleus is occupied by out-of-focus images of colored constituents in the figure, but is really devoid of stained material. Technique as in Fig. 7. •~1,800. 454 S. Takagi, J. Kitada, H. Masuda and M. Tagawa Cytologia 26 intensely in both kinds of cell. Resorcin-fuchsin does not stain any definite structure in the cytoplasm of the cells examined. Staining with sudan black reveals lipid droplets colored blue to black in the cytoplasm of the fibroblasts. In the Golgi area, lightly stained filament ous elements are found, which form a dense agglomerate in some cells. The mitochondria are also lightly stained by the dye. In some cells, tiny lipid droplets are found between the filaments in the Golgi area, giving an im pression that they make their first appearance there, increase in size and migrate toward the cell periphery. In the tumor cells, larger lipid droplets in the cytoplasm are intensely stained by sudan black and beside the nucleus an area is left where no lipid droplets except small ones are found. Hence this area is lighter than the rest of the cytoplasm and includes Golgi filaments that are faintly stained with the dye. In the peripheral cytoplasm, besides the lipid droplets and the mitochondria which are also stained, the elements of the endoplasmic reticulum seem to take up the dye (Fig. 6). After the tumor cells are treated with trypsin, their Golgi filaments appear to be stained with sudan black more intensely than otherwise. When the fibroblasts are fixed in calcium-formol and stained with Nile blue, the filaments in the Golgi area are stained light blue and the mit ochondria in the peripheral cytoplasm are stained distinctly blue. In the similarly treated tumor cells, Golgi filaments, mitochondria and lipid droplets are stained of a violet color. This is the same when the materials are sub jected to trypsin pretreatment. In the two kinds of cells, lipid droplets and mitochondria show a positive reaction to the acid hematein test, which does not occur after pyridine ex traction, thus indicating the presence of a phospholipid. The Golgi filaments always react negatively to the test. Fine filamentous Golgi elements with blurred contour are stained only faintly in the periodic acid-Schiff reaction in both types of cells, whereas the mitochondria are distinctly colored. It seems that the elements of the endoplasmic reticulum are also stained. Lipid droplets are not reactive (Figs. 7, 8). The result is essentially the same after the treatment with amylase, though the staining is generally reduced in intensity. Alkaline phosphatase is not localized in any definite part of the cytoplasm in the two types of cells.

Discussion

Among the results obtained, the followings seem to be the most im portant: (1) The Golgi complex under the phase microscope consists of canalicules and filaments and ground substance. The canalicules are thought to represent dilatations of the filaments and are lacking in the ascites tumor cells. (2) The Golgi filaments are more slender than the mitochondria and 1961 Staining and Histochemical Reactions of Golgi Com plex 455

are blurred in contour . (3) The Golgi filaments and the ground substance are darkened by the addition of osmium to the living cell . (4) The Golgi fi laments and the ground substance are stained with Altmann's acid f uchsin i n Champy fixed cells. (5) The Golgi filaments are vitally stained with Janus green like the mitochondria. Thus the Golgi filaments have several properties in common with mito chondria. Vital staining with Janus green and staining with acid fuchsin in histological sections have been regarded as a useful means by which to identify mitochondria. The Golgi filaments can , however, be discriminated from the mitochondria by the following characteristics . (1) Their juxta nuclear position. (2) They are blackened more readily by osmium tetroxide than mitochondria. (3) For vital staining , they require a higher concentra tion of Janus green than mitochondria . (4) They are threads finer than mitochondria and possess a blurred contour . It can hardly be doubted that the Golgi filaments have often been con fused with mitochohdria. For example , in reading the following passage, we cannot but consider that at least part of the "mitochondria" around the cytocentrum may in reality be Golgi filaments . (In the fibroblasts) "the mitochondria appear as slender rods which are scarce in the processes , but more numerous near the nucleus and around the cytocentrum" (Maximow and Bloom 1957). Ludford (1951) found an aggregation of filaments in this part of the cytoplasm of cultured fibroblasts and interpreted them as degenerating or ill-defined mitochondria. It is evident on inspecting his photomicrographs that they correspond to the Golgi canalicules and filaments as we have described above. Recently, King and Roe (1956) stained "mitochondria" of the ascites tumor cell in a concentrated solution of Janus green B and found them in the "granular zone" adjacent to, and in the indentation of, the nucleus. Since we have found veritable mitochondria in the cytoplasm outside of his "granular zone", we think it very probable that what he could stain with Janus green were Golgi filaments . It has been stated by the previous investigators that the elements of Golgi complex are vitally stainable by other dyes than those we have em ployed. They are stained, for example, with Nile blue (Tanaka 1933, in cultured nervous cells and retina cells; Tarao 1953, in mouse nerve cells and newt hepatic cells) and with chrysoidine and Nile blue (Dalton and Felix 1954, Golgi substance isolated from epididymis). It is cited in recent reviews by Bourne (1955) and Cowdry (1955), that the Golgi elements in fixed cells are occasionally stained with resorcin-fuchsin and iron hematoxylin, showing that protein is one of their constituents, and also that they are stained with sudan black, which indicates, as well as the blackening with osmium tetroxide, the presence of a lipid. The presence of vitamin C in the Golgi complex has often been emphasized. A number of recent investigations, including Martin's (1958) and Allen and Slater's (1958), make it almost indubitable 456 S. Takagi, J. Kitada, H. Masuda and M. Tagawa Cytologia 26

that alkaline and acid phosphatases are localized in this organelle in some types of cells. Although Lacy (1954) mentioned that the lipid of the Golgi apparatus in the pancreatic cells is stainable with sudan black only after unmasking by means of silver nitrate, Nagatani (1956) made it clear that the organelle in the newt liver cells can be stained with sudan black and acid hematein in either masked or unmasked condition. By dint of the studies by Gersh (1949) and Schneider and Kuff (1954) we come to believe that the Golgi apparatus in some tissues contains a PAS-positive material. It may be an interesting, but difficult, question, whether the staining with dyes and the reactions indicating the presence of enzymes or other substances take place in the membranes delimiting the Golgi canalicules and filaments or within their interior. The contents of the Golgi canalicules have been considered to be a watery solution (Bensley 1951), a conclusion which is supported on evidences of electron microscopic study (Gatenby and Lufty 1956) and of refractometry and microincineration (Kruszynski 1957). There is, however, considerable evidence that the substances absorbed into, or secreted from the cell are temporarily stored in the Golgi canalicules or vacuoles. Lipid droplets are taken by pinocytosis into the cytoplasm and are enclosed within the Golgi vacuoles in the columnar epithelial cells of the intestine (Palay and Karlin 1959). In secreting cells, Golgi membranes enclose maturing or matured secretion granules (Farquhar and Wellings 1957, Palay 1958, Hirsch 1960). Hence, it is possible that the Golgi canalicules and filaments include these substances in their internal cavities and give histochemical reactions which may vary according to their contents. However, the reduction of osmium tetroxide and the staining with Janus green and the other vital dyes, which we have described in the present re port, are believed to have taken place in the membranes delimiting the Golgi canalicules and filaments. For, as we have stated in the previous report (Takagi and Masuda 1956), the blackening and the staining take place in the surrounding membranes of the Golgi canalicules, in the beginning at least, and not in their interior. Palade (1955) considers that the Golgi complex represents a local dif ferentiation of the endoplasmic reticulum and, indeed, evidences of continuity between the two have been presented by many investigators. However, on evidences from the present study the following proposition seems to be established that the Golgi complex, situated always on the side of the nucleus, differs from the endoplasmic sacs that may be distributed widely in the other parts of the cytoplasm, not only in its morphological characteristics, but also in its stainability towards dyes, and in this respect the Golgi complex re sembles mitochondria rather than the endoplasmic reticulum. The ground substance of the Golgi area turns gray with osmium tetrox ide and is stainable with acid fuchsin and trimethylthionin. It is often darker than the remaining cytoplasm with phase optics. This feature of the 1961 Stainingand HistochemicalReactions of GolgiComplex 457

Golgi ground substance may be attributed to the presence of piles of fine granules or vesicles, which are recognized by electron microscopic studies, but are not resolved with the optical microscope.

Summary 1. Several staining and histochemical properties of the components of Golgi complex of the fibroblasts cultured in vitro and the ascites tumor cells of the mouse were studied. 2. When examined with phase optics, the Golgi complex of the living fibroblast consists of canalicules and filaments, while that of the tumor cell is composed only of filaments. The filaments are finer than the mitochondria and blurred in outline. Since the walls of canalicules show the same stain ing and histochemical properties with the filaments, the canalicules are thought to belong to a category of filaments, which are either dilated locally or are dilated whole-length. 3. The Golgi filaments (the word is used in the broad sense, as in dicated above, in this and the following sections) are blackened more readily than mitochondria by the addition of osmium tetroxide. They are stained red with Altmann's acid fuchsin as mitochondria. 4. Janus green stains the Golgi filaments as it does mitochondria. In the fibroblast, a higher concentration of the dye is evidently required to stain the Golgi filaments than mitochondria. 5. The Golgi filaments visualized by blackening with osmium, by stain ing with acid fuchsin and by vital staining with Janus green are distinguished from mitochondria, as the former are more slender and less smooth in outline than the latter and are possessed of a characteristic of gathering in the vicinity of the nucleus. 6. The Golgi filaments are stained vitally with neutral red, and vitally and metachromatically with methylene blue, trimethylthionin and toluidine blue. 7. The ground substance of the Golgi area is often darker or lighter under the phase microscope than the remainder of the cytoplasm. It be comes darkened with osmium and is stained post mortem with acid fuchsin and intra vitam with trimethylthionin. 8. The Golgi filaments in the both types of cells are not stained with iron hematoxylin and resorcin-fuchsin. They do not react positively to the acid hematein test, nor to the periodic acid-Schiff and alkaline phosphatase techniques. They are weakly stained with sudan black and Nile blue in the calcium-formol fixed materials. 458 S. Takagi, J. Kitada, H. Masuda and M. Tagawa Cytologia 26

References Allen, J. and Slater, J. J. 1958. A chemical and histochemical study of acid phosphatase in the epididymis of normal, castrate and hormone replaced castrated mice. Anat. Rec. 130: 731-745. Bensley, R. R. 1951. Facts versus artefacts in cytology: The Golgi apparatus. Exptl. Cell Research 2: 1-9. Bourne, G. H. 1954. Some chemical and biochemicalaspects of the Golgi apparatus. Jour. Roy. Micros. Soc. 74: 180-187. Cowdry, E. V. 1955. In "Cancer Cells". Philadelphia and London: W. B. Saunders Co. Dalton, A. J. and Felix, M. D. 1954. Cytologic and cytochemicalcharacteristics of the Golgi substance of epithelial cells of the epididymis-in situ, in homogenates and after isolation. Am. J. Anat. 94: 171-207. - 1956. The electron microscopy of normal and malignant cells. Ann. N. Y. Acad. Sci. 63: 1117-1140. - 1957. Electron microscopy of mitochondria and the Golgi complex. Symposia of the Soc. for Exp. Biol. 10: 148-159. Farquhar, M. G. and Wellings, S. R. 1957. Electron microscopic evidence suggesting secre tory granule formation within the Golgi apparatus. J. Biophysic. and Biochem. Cytol. 3: 319-322. Gatenby, J. B. and Lufty, R. G. 1956. The Golgi apparatus and the electron microscope. Nature 177: 1027-1029. Gersh, I. 1949. A protein component of the Golgi apparatus. Arch. Path. 47: 99-109. Haguenau, F. et. Bernhard, W. 1955. L'appareil de Golgi dans les cellules normales et cancereuses de vertebras. Arch. d'anat. microsc. 44: 27-55. Hirsch, G. C. 1960. Die Fliessbandarbeit in der exokrinen Pankreaszelle bei der Produktion von Enzymen. Mit einem Exkurs uber Ergastoplasma and Golgi-Korper. Naturwiss. 47: 25-35. King, R. J. and Roe, E. M. F. 1956. A study of fresh ascites tumor cells by ultraviolet, phase-contrast and interference microscopy. Jour. Roy. Micros. Soc. 76: 168-174. Kruszynski, J. 1957. Golgi structure of adenopituitary cells revealed by refractometry and microincineration. Exptl. Cell Research 13: 189-192. Lacy, D. 1954. Chemical composition of the Golgi apparatus in the exocrine and endocrine cell in the pancreas of the mouse. Nature 173: 1235. - 1957. The Golgi apparatus in neurons and epithelial cells of the common limpet Patella vulgata. J. Biophysic. and Biochem. Cytol. 3: 779-796. Ludford, R. J. 1951. Pathological aspects of cytology, In "Cytology and Cell Physiology" 2nd Ed., Oxford: the Clarendon Press. Martin, B. F. 1958. Histological and histochemical studies on the bladder and ureter, with particular reference to alkaline phosphatase and Golgi material. J. Anat. 92: 286-297. Maximow, A. A. and Bloom, W. 1957. "A Textbook of ". 7th Ed. Philadelphia and London: W. B. Saunders Co. Nagatani, Y. 1956. Staining of cytoplasmic granules by carcinogenic azo-dye, o-amino-azo toluene. Sci. Rep. Tohoku Univ. 4th Ser. 22: 207-214. Palade, G. E. 1955. Studies on the endoplasmic reticulum. II. Simple dispositions in cells in situ. J. Biophysic. and Biochem. Cytol. 1: 567-582. Palay, S. L. 1958. The morphology of secretion. In "Frontiers in Cytology". New Haven: Yale University Press. - and Karlin, L. J. 1959. An electron microscopic study of the intestinal villus. I. The fasting animal. J. Biophysic. and Biochem. Cytol. 5: 363-372. Schneider, W. C. and Kuff, E. L. 1954. On the isolation and some biochemical properties 1961 Staining and Histochemical Reactions of Golgi Complex 459

of the Golgi substance. Am. Anat. 94: 209-224. Sjostrand, F. S. 1956. The ultrastructure of cells as revealed by the electron microscope. Intern. Rev. of Cytol. 5: 455-533. Takagi, S. 1958. Studies of the Golgi complex in the living cell. Jap. J. Exptl. Morph. 12: 1-11. - and Masuda , H. 1956. Fine structure of the Golgi apparatus of the fibroblasts in vitro as revealed by phase microscopy and vital staining. Bull. Univ. of Osaka Pref., Ser. B 6: 33-41. - and Tagawa , M. 1957. The Golgi apparatus of frog lymphocytes as revealed by phase microscopy and vital staining. Bull. Univ. of Osaka Pref., Ser. B 7: 9-13. Tanaka, H. 1933. Golgi apparatus vitally stained with Nile-blue sulphate. Arch. exper. Zellforsch. 13: 48-52. Tarao, S. 1939. Microchemical studies on the Golgi apparatus using protease-Nile blue sulphate technique I. Golgi apparatus of hepatic cells in the mouse and the newt. J. Fac. Sci., Hokkaido Imp. Univ., Ser. 6 7: 1-16. - 1953. Vital staining of the Golgi apparatus. Cytologia 18: 218-228. Weiss, J. M. 1955. The role of the Golgi complex in fat absorption as studied with the electron microscope with observations on the cytology of duodenal absorptive cells. J. Exptl. Med. 102: 775-782.