Morphology and histochemistry of the adrenal medulla. I. Various types of primary catecholamine-storing cells in the mouse adrenal medulla.

Semithin sections (Araldite) of mouse adreno-medullary tissue were examined in the light microscope after perfusion fixation with glutaraldehyde, glutaraldehyde/formaldehyde or after freeze-drying followed by a treatment with hot formaldehyde gas. The following methods were employed: (i) aldehyde-induced fluorescence of catecholamines, (ii) Schmorl's ferric ferricyanide reaction, (iii) argentaffin reaction, and (iiii) staining with alkaline lead citrate followed by Timm's silver sulphide reaction. The correspondence of results obtained by the various methods was proven in consecutive sections or by successively applying different methods to identical sections. Four types of primary catecholamine-storing cells were identified. NA1 cells contain cytoplasmic granules up to 0.3 mum in diameter which stain black with ammoniacal silver and display a bright white to yellow fluorescence. NA2 cells show smaller cytoplasmic granules which stain brown with the argentaffin method and give white catecholamine fluorescence. NA3 cells appear yellow-earth after applying the argentaffin reaction and show greenish fluorescence. NA4 cells are hardly identified in the light microscope. These cells are significantly smaller than the above mentioned cells and characterized by a high nucleo-cytoplasmic ratio. They become straw coloured with ammoniacal silver and show greenish fluorescence. The argentaffin reaction was also used to identify these cells in semithin sections of glutaraldehyde/osmium tetroxide fixed material. The fine structure of the various noradrenalin-storing cells was studied in consecutive thin sections. NA1 cells were found to contain two populations of granules, the larger ones measuring between 300 and 350 nm, the smaller ones about 175 nm. The granules in NA2 cells correspond to this latter population (175 nm). NA3 cells contain an uniform granule population with a main diameter of 120 nm. The smallest granules are seen in NA4 cells being in the dimension of 80 nm. Granules in NA1 and NA2 cells show uniformly high density, whereas those in NA3 and NA4 cells display cores of varying density. Granules with moderately dense cores in NA3 and NA4 cells may represent partially emptied sites of noradrenalin storage or dopamin containing particles.     

A simple impregnation technique for thin and semithin enterochromaffin cells in sections

Summary Constant, intense and precise impregnation of enterochromaffin (EC) cells was achieved simply by floating thin or semithin sections of gut mucosa, fixed in osmium tetroxide or in glutaraldehyde with postfixation in osmium, on a silver nitrate or proteinate solution. EC cells alone showed impregnation in the light microscope. In the electron microscope, impregnation affected not only the secretory granules of EC cells but also, although much more faintly, those of other, non-EC cells (D, X, D₁, G and other cells). Lysosomes also showed partial or total reactivity. Oxidation reduced but did not entirely suppress EC cell staining and had no effect on non-EC endocrine cell staining. Since the reaction did not occur with glutaraldehyde alone, osmium appeared to be a crucial component of the process. These findings should be borne in mind in applying Thiery's method for vicinal glycol groups to the type of study material used in these experiments.     

A simple impregnation technique for thin and semithin enterochromaffin cells in sections

Constant, intense and precise impregnation of enterochromaffin (EC) cells was achieved simply by floating thin or semithin sections of gut mucosa, fixed in osmium tetroxide or in glutaraldehyde with postfixation in osmium, on a silver nitrate or proteinate solution. EC cells alone showed impregnation in the light microscope. In the electron microscope, impregnation affected not only the secretory granules of EC cells but also, although much more faintly, those of other, non-EC cells (D, X, D1, G and other cells). Lysosomes also showed partial or total reactivity. Oxidation reduced but did not entirely suppress EC cell staining and had no effect on non-EC endocrine cell staining. Since the reaction did not occur with glutaraldehyde alone, osmium appeared to be a crucial component of the process. These findings should be borne in mind in applying Thiery's method for vicinal glycol groups to the type of study material used in these experiments.     

SGC (small granule chromaffin) cells in the mouse adrenal medulla: light and electron microscopic identification using semi-thin and ultra-thin sections.

Adrenal glands of the mouse, fixed either in glutaraldehyde followed by osmium tetroxide or in a mixture of potassium dichromate and glutaraldehyde, and embedded in Epon 812, were investigated by light and electron microscopy. An argentaffin reaction was applied to semi-thin sections for light microscopy and to ultra-thin sections for electron microscopy. Since the mature secretory granules in the Small Granule Chromaffin (SGC) cell were argentaffin and were mainly located along the cell membrane, this cell was clearly distinguishable under the light microscope both from the A (adrenaline) cell whose secretory granules were non-argentaffin and from the NA (noradrenaline) cell whose cytoplasm was rich and was filled with large, strongly argentaffin granules. Chromaffinity of the SGC cell was demonstrated under the light microscope. The SGC cell was intensively stained with toluidine blue without revealing metachromasia. It was demonstrated at the EM level that not only the secretory granules but also the synaptic-like vesicles in the SGC cell contained argentaffin substances. Possible functional relationship between the secretory granules and the synaptic-like vesicles was discussed.     

Electron microscopic classification of amine-producing endocrine cells by selective staining of ultra-thin sections

Summary The argyrophil, argentaffin and chromaffin reactions were performed directly on ultra-thin sections for examination in the electron microscope. Glutaraldehyde fixation was appropriate for the argentaffin and chromaffin reactions; additional fixation with osmium tetroxide, however, caused impairment of these reactions. Fixation with formaldehyde, but not with glutaraldehyde, was adequate for the argyrophil reaction; post-fixation with osmium tetroxide did not affect this staining. At the light microscopic level the staining reactions were correlated with fluorescence histochemistry according to the method of Falck and Hillarp. The techniques described were used to study certain amine-producing endocrine cell systems: adrenal medullary cells and thyroid parafollicular cells of the mouse, gastric endocrine cells from the oxyntic gland area of the mouse, rat and rabbit. All these cells stained argyrophil. The adrenal medullary cells and one cell type in the oxyntic gland area of the rabbit were strongly argentaffin and chromaffin. The remainder of the cells were non-argentaffin and non-chromaffin but could be induced to give an argentaffin (and chromaffin) reaction after injection of the animals with l-3,4-dihydroxyphenylalanine or l-5-hydroxytryptophan, a treatment which is known to result in the accumulation of the highly reducing dopamine and 5-hydroxytryptamine, respectively, in these endocrine cells. Without exception the precipitates formed in all the staining reactions accumulated selectively over the secretory granules of the cells.The techniques described permit differential staining of consecutive ultra-thin sections for electron microscopic characterization of one and the same cell. They will provide information necessary for correlative studies of the stainable cells at the light and electron microscopic levels.     

Application of silver stains to gastric endocrine cells in plastic sections

Summary A simultaneous light and electron microscopic study of mouse gastric mucosa was made to determine whether the silver nitrate methenamine stain of Duk-Ho Lee could be used to stain gastric endocrine-like cells in plastic embedded tissue. Examination of consecutive thick and thin sections showed that this stain blackened the granules of the predominant type of endocrine-like cell present. Blackening of the granules with silver occured in tissue fixed in osmium tetroxide solution with or without dichromate salt or in tissue fixed in glutaraldehyde then treated with osmium. The intensity of staining was deepest in the osmium-dichromate fixed tissue, but the glutaraldehyde-osmium procedure gave less interference from diffuse silver impregnation and better preservation of detail for electron microscopy.     

Ultrastructural cytochemistry of the secretory granules of the hamster submandibular gland

Summary The ultrastructure and cytochemistry of the secretory granules of the male hamster submandibular salivary gland were studied. After fixation in glutaraldehyde followed by osmium tetroxide the granules exhibit a characteristic bipartite substructure, with an electron lucid crescenteric rim and a more dense central core. A differentiation into two regions of the granules could also be visualized in specimens primarily fixed in Millonig's osmium tetroxide or in potassium permanganate. The electron lucid peripheral portion of the membrane bounded secretory granules further displays a strong positive reaction after staining of ultrathin sections with the periodic acid-chromic acid-(PA-CrA)-silver technique. The strong periodate reactivity of the rim relative to the core, suggests a difference in mucin composition of the two granule regions. With the PA-CrA-silver staining technique a positive reaction was also observed within the Golgi apparatus of the acinar cells.     

SILVER IMPREGNATION OF ULTRATHIN SECTIONS FOR ELECTRON MICROSCOPY

A new procedure is described for silver impregnation of thin sections for electron microscopy. Sections of various tissues, fixed in OsO₄ and embedded in methacrylate, were treated with an ammoniacal silver solution, directly or after oxidation with periodic acid or hydrogen peroxide. After OsO₄ fixation all cellular membranous systems exhibit a primary argentaffinity probably due to the reduction of ammoniacal silver solution by the reduced osmium bound to unsaturated lipids. Bleaching the sections with hydrogen peroxide removes the argentaffinity of protoplasmic structures. Treatment of the sections with periodic acid results in decreased argentaffinity of protoplasmic components while the argentaffinity of metaplasmic structures is greatly enhanced. The latter procedure appears particularly useful for enhancing the contrast of basement membranes.     

Imidazole-buffered osmium tetroxide: an excellent stain for visualization of lipids in transmission electron microscopy

Summary The usefulness of imidazole-buffered osmium tetroxide as a stain for lipids in transmission electron microscopy has been investigated. Rat liver and other tissues were fixed by perfusion with glutaraldehyde and post-fixed with osmium-imidazole and the appearance of lipid droplets was compared with that after post-fixation in unbuffered aqueous osmium tetroxide or an osmium solution buffered otherwise. Prominent electron-opaque staining of lipid droplets and of lipoprotein particles was noted after post-fixation with 2% osmium-imidazole, pH 7.5, for 30 min. The lipid droplets appeared well circumscribed with no evidence of diffusion. In contrast, the intensity of staining was much less and there was some diffusion around lipid droplets in material post-fixed in aqueous or cacodylate-buffered osmium tetroxide. Spot tests on filter paper revealed that unsaturated fatty acids, especially linolenic and linoleic acids reacted more intensely with osmium-imidazole than with aqueous osmium tetroxide. These findings demonstrate that osmium-imidazole provides an excellent stain for lipids in transmission electron microscopy and that most probably it stains lipids with unsaturated fatty acids.     

Lipid staining for the electron microscope: a new method.

Tissues fixed in osmium tetroxide or in combined osmium and glutaraldehyde (Hinde), embedded in Spurr's medium, cut at 0-5-I mum and mounted in Farrants' gum medium containing ethyl gallate, show good staining of lipid-contaning structures (droplets of triglyceride, membranes, mitochondria, etc.) in the light microscope. Such preparations show moderate contrast in the electron microscope without further staining. But a specific increase in contrast in lipid-rich structures is obtained by partition of the tissues, before embedding, in 70% ethanol saturated with the monoterpene hydrocarbon myrcene, with or without the addition of 0-I % ethyl gallate, followed by osmium tetroxide. This method will visualize both saturated and unsaturated lipids, including waxes.     

Morphology and histochemistry of the adrenal medulla

Summary Semithin sections (Araldite) of mouse adreno-medullary tissue were examined in the light microscope after perfusion fixation with glutaraldehyde, glutaraldehyde/formaldehyde or after freeze-drying followed by a treatment with hot formaldehyde gas. The following methods were employed: (i) aldehyde-induced fluorescence of catecholamines, (ii) Schmorl's ferric ferricyanide reaction, (iii) argentaffin reaction, and (iiii) staining with alkaline lead citrate followed by Timm's silver sulphide reaction. The correspondence of results obtained by the various methods was proven in consecutive sections or by successively applying different methods to identical sections.Four types of primary catecholamine-storing cells were identified. NA₁ cells contain cytoplasmic granules up to 0.3 m in diameter which stain black with ammoniacal silver and display a bright white to yellow fluorescence. NA₂ cells show smaller cytoplasmic granules which stain brown with the argentaffin method and give white catecholamine fluorescence. NA₃ cells appear yellow-earth after applying the argentaffin reaction and show greenish fluorescence. NA₄ cells are hardly identified in the light microscope. These cells are significantly smaller than the above mentioned cells and characterized by a high nucleo-cytoplasmic ratio. They become straw coloured with ammoniacal silver and show greenish fluorescence.The argentaffin reaction was also used to identify these cells in semithin sections of glutaraldehyde/osmium tetroxide fixed material. The fine structure of the various noradrenalin-storing cells was studied in consecutive thin sections. NA₁ cells were found to contain two populations of granules, the larger ones measuring between 300 and 350 nm, the smaller ones about 175 nm. The granules in NA₂ cells correspond to this latter population (175 nm). NA₃ cells contain an uniform granule population with a main diameter of 120 nm. The smallest granules are seen in NA₄ cells being in the dimension of 80 nm. Granules in NA₁ and NA₂ cells show uniformly high electron density, whereas those in NA₃ and NA₄ cells display cores of varying density. Granules with moderately dense cores in NA₃ and NA₄ cells may represent partially emptied sites of noradrenalin storage or dopamin containing particles.Supported by the Deutsche Forschungsgemeinschaft, grant Nr. Bo 525/1These results were presented in part at 17. Tagung der Deutschen Gesellschaft für Elektronenmikroskopie, Berlin, 21.–26. 9. 1975     

Light microscopic localization of cytochemical reactions in epoxy-embedded material for electron microscopy.

Tissues from mice were fixed in 1.5% glutaraldehyde, treated for the ultrastructural localization of alkaline phosphatase or Mg++-dependent adenosine triphosphatase, post-fixed in osmium tetroxide, dehydrated and embedded in plastic for electron microscopy. The sites of reaction were visualized in 1-mu plastic sections counterstained with toluidine blue, using a phase contrast microscope. The data show a close correlation between the sites of reaction observed with the phase contrast microscope and the sites studied with the electron microscope. The use of this technique for the study of these phosphatases in normal and pathologic tissues is recommended in order to achieve a high degree of accuracy in selecting a portion of the tissue sample for electron microscopy and to obtain greater resolution in the localization of these enzymes with the light microscope.     

HISTOLOGICAL STAINING OF LIPIDS FOR THE LIGHT AND ELECTRON MICROSCOPE

1. It is generally agreed that the blackening of osmium tetroxide by unsaturated lipid is too unpredictable to demonstrate lipid in tissues.
2. At neutral pH osmium tetroxide combines with the double bonds in the lipoproteins of cellular membranes (mitochondria, etc.) and the deep colour reaction of ethyl gallate with this osmium provides good staining of lipid for the light microscope.
3. Osmium taken up by tissue proteins at neutral pH is only a small fraction of that taken up by the lipid. (After acid fixatives osmium tetroxide is a general protein stain.)
4. The uptake of Sudan black B by partition from dilute solution is a specific test for lipid, but in normally fixed tissue most of the structural lipid is 'bound' and is not accessible to the dye.
5. Cautious treatment of fixed tissue with dilute sodium hypochlorite will unmask this lipid for viewing by the light microscope.
6. Direct fixation with neutral osmium tetroxide is an effective method for visualizing lipid for the electron microscope (as in the ethyl gallate method for the light microscope). But the poor penetration of osmium limits its use in this way.
7. After formol/glutaraldehyde fixation much of the lipid in the tissues is 'bound' and does not take up osmium. It can be unmasked by a saturated aqueous solution of thymol.
8. The unmasked lipid can then be rendered more osmiophil by partition in a solution of the highly unsaturated terpene farnesol, thus increasing the uptake of osmium in a renewed application.
9. Some of the novel observations on tissue lipids made by these methods are reviewed.     

Cytochemical localization of catalase in leaf microbodies (peroxisomes)

Segments of mature tobacco leaves were fixed in glutaraldehyde, incubated in medium containing 3,3''-diaminobenzidine (DAB) and hydrogen peroxide, and postfixed in osmium tetroxide. Electron microscopic observation of treated tissues revealed pronounced deposition of a highly electron-opaque material in microbodies but not in other organelles. The coarsely granular reaction product is presumably osmium black formed by reaction of oxidized DAB with osmium tetroxide. Reaction of the microbodies with DAB was completely inhibited by 0.02 M 3-amino-1,2,4-triazole and was considerably reduced by 0.01 M potassium cyanide. These results, when considered in light of recent biochemical studies, strongly suggest that catalase is responsible for the reaction. Sharp localization of this enzyme in microbodies establishes that they are identical to the catalase-rich "peroxisomes" recently isolated from leaf cell homogenates. A browning reaction that occurred in leaves during the incubation step was inhibited by cyanide but not by aminotriazole and therefore could not have been caused by the same enzyme. This reaction and a slight deposition of dense material within primary and secondary walls are ascribed to oxidation of DAB by soluble and wall-localized peroxidases.     

THE LOCALIZATION OF ACID PHOSPHATASE IN RAT LIVER CELLS AS REVEALED BY COMBINED CYTOCHEMICAL STAINING AND ELECTRON MICROSCOPY

Discrete localization of stain in pericanalicular granules was found in 10 µ frozen sections of formol-phosphate-sucrose-fixed liver stained by the Gomori acid phosphatase technique and examined in the light microscope. The staining patterns, before and after treatment with Triton X-100 and lecithinase, were identical with those previously reported for formol-calcium-fixed material treated in the same way, and it can be assumed that the stained granules are identical with "lysosomes." Examination in the light microscope of the staining patterns and lead penetration in fixed blocks and slices of various dimensions showed nuclear staining and other artefacts to be present, produced by the different rates of penetration of the various components of the staining medium into the tissue. A uniform pericanalicular staining pattern could be obtained, however, with slices not more than 50 µ thick, into which the staining medium could penetrate rapidly from both faces. The staining pattern produced in 50 µ slices was the same both at pH 5.0 and pH 6.2, and was not altered by subsequent embedding of the stained material in butyl methacrylate. Electron microscopy showed the fine structure of fixed 50 µ frozen slices to be well preserved, but it deteriorated badly when they were incubated in the normal Gomori medium at pH 5.0 before postfixing in osmium tetroxide. After incubation in the Gomori medium at pH 6.2, the detailed morphology was substantially maintained. In both cases lead phosphate, the reaction product, was found in the pericanalicular regions of the cell, but only in the vacuolated dense bodies and never in the microbodies. Not every vacuolated dense body contained lead, and stained and unstained bodies were sometimes seen adjacent to each other. This heterogeneous distribution of stain within a morphologically homogeneous group of particles is consistent with de Duve's suggestion (9) that there is a heterogeneous distribution of enzymes within the lysosome population. It is concluded from these investigations that the vacuolated dense bodies seen in the electron microscope are the morphological counterparts of the "lysosomes" defined biochemically by de Duve.     

A Golgi-electron microscope method for insect nervous tissue.

Golgi's light microscope method of selective silver impregnation for nervous tissue combined with electron microscopy appears to offer a promising method for working out the detailed anatomy of individual neurons and their connections. Insect nervous tissue is fixed in a mixture of 2% paraformaldehyde and 2 1/2% glutaraldehyde in Millonig's buffer (pH 7.2) before postfixation for 12 hours in a solution brought to pH 7.2 with KOH containing 2% potassium dichromate, 1% osmium tetroxide and 2% D-glucose. The tissue is then transferred to a solution of 4% potassium dichromate for 1 day; and for 1-2 days to a 0.75% silver nitrate solution. After dehydration and embedding in Araldite, 50 mum sections are made. Areas of interest are cut from these sections and re-embedded in silicone molds. Ultrathin sections are then cut and stained with uranyl acetate and lead citrate. The Golgi method described here gives good results at the level of both light and electron microscopy.     

Ultrastructural identification of argyrophil and argentaffin cells of the gastric mucosa in the rat

Ultrastructure of endocrine cells impregnated in the rat gastric mucosa by Grimelius method (identification of argyrophilia) and by Masson--Hamperl method (identification of argentaffinity) and influence of various fixatives on the structure and properties of the secretory granules in these cells have been studied. Fixation of the material in paraformaldehyde or glutaraldehyde varies in its effect on the granule structure of EC-, D1- and ECL-cells, while its influence on the granule structure of G-, D- and AL-cells is identical. The granules of EC-, ECL- and G-cells are argyrophil, and only those of EC-cells are argentaffin. Weak argyrophilia, which is evidently not appearant at the light-optical level, is specific for granules of D1- and AL-cells. Fixation in paraformaldehyde and especially the subsequent treatment in osmium tetroxide results in increasing argyrophilia of the endocrine cells, as compared to fixation in glutaraldehyde. Varieties in the effect of the fixatives do not prevent ultrastructural and histochemical identification of the endocrine cell types.     

The fine structure of the oxytalan fiber

Maraglas-embedded sections of periodontal membranes around continually growing incisors of Sprague-Dawley rats fixed consecutively in cold glutaraldehyde and Palade's 1 % buffered osmium tetroxide were examined under the electron microscope for oxytalan connective tissue fibers. Oxytalan fibers were noted to consist of bundles of filaments approximately 150 A in diameter with an interfilamentous amorphous substance of approximately the same diameter. A periodicity of fibrillar elements was not obvious.     

Localization of the synaptonemal complex under the light microscope

The use of osmium tetroxide fixation followed by postreatment with p-phenylenediamine gives an opportunity of locating the synaptonemal complex (SC) under the light microscope in mouse testes and Allium cepa anthers. When semi-thin sections from these materials were observed under phase contrast optics or dark field microscopy, fine threads in the pachytene nuclei were clearly visible. Post-staining of semi-thin sections with ammoniacal silver increased the contrast of the SC and allowed for observations using a bright field illumination. Ultrathin sections of osmium tetroxide/ p-phenylenediamine treated material showed that, under the electron microscope, this technique stains preferentially elements of the synaptonemal complex, while the surrounding chromatin remains unstained.     

Osmolarity of osmium tetroxide and glutaraldehyde fixatives

Synopsis The evidence available to date for the importance of fixative osmolarity is considered together with some observations on the volume changes of crab axons after fixation by osmium tetroxide and glutaraldehyde. The results obtained are compared with those obtained from crab axons and from amphioxus skin cells which had been processed and examined with the electron microscope after initial fixation in fixatives of different composition. It is concluded that the osmolarity of the fixative vehicle is of considerable importance when the fixing agent is glutaraldehyde but is of less importance when the fixing agent is osmium tetroxide or a mixture of the two agents.Preliminary observations upon crab axons fixed with glutaraldehyde in a vehicle approximating to the internal composition of the cells suggest that this approach to the design of fixative vehicles may be useful.