THE DISTRIBUTION OF INORGANIC CATIONS IN MOUSE TESTIS : Electron Microscope and Microprobe Analysis

For localization of pyroantimonate-precipitable cations, mouse testes were fixed with a saturated aqueous solution of potassium pyroantimonate (pH about 9.2, without addition of any conventional fixative), hardened with formaldehyde, and postosmicated. A good preservation of the cell membranes and over-all cell morphology is obtained as well as a consistent and reproducible localization of the insoluble antimonate salts of magnesium, calcium, and sodium. Four sites of prominent antimonate deposits are revealed, besides a more or less uniform distribution of the precipitates. These sites are: (a) In the walls of the seminiferous tubules, localized in two concentric layers corresponding to the inner and outer layers of the tubular wall; (b) Around the blood vessels and adjacent connective tissue; (c) At the area of contact between the Sertoli cell and spermatids, where a double line of precipitate surrounds the head of the mature spermatids; and (d) In the cell nuclei, disposed between regions of the condensed chromatin. The nucleus of mature spermatids did not show any sign of antimonate precipitation. The implications of this inorganic cation distribution are discussed with relation to their anionic counterparts, their localization in other animal and plant tissues, and the possibility that those sites may represent barriers to the free passage of ions.     

Troponin bound calcium in electron micrographs of myocardial cells as demonstrated by the potassium-antimonate technique

Summary Following the K-antimonate reaction in atrial myocardial tissue, a pattern of evenly spaced cross striations of antimonate precipitates is demonstrated along the myofilaments. This spacing, found in both turtle and mouse atria, has a periodicity of about 400 Å. In order to test the shifts of the antimonate reaction product in the tissue, a comparison is made between the localization of the antimonate precipitate as seen in viz. thin plastic sections and in cryo-ultra sections being dry-cut at -90° C from N₂ frozen tissue. Preliminary results suggest only minor distributional differences in the sarcomeric pattern. On the basis of these tests, and, on the basis of previous studies by means of X-ray microanalysis, it is suggested that the periodic pattern of evenly spaced precipitates, reflects the localization of troponin bound calcium along the thin filaments during contraction.This work was supported by grants from The Norwegian Research Council for Science and the Humanities. We are also indebted to Mrs. Trine Jensen and Miss Sigrid Devik for skilful technical assistance.     

THE INTRACELLULAR LOCALIZATION OF INORGANIC CATIONS WITH POTASSIUM PYROANTIMONATE : Electron Microscope and Microprobe Analysis

Potassium pyroantimonate, when used as fixative (saturated or half-saturated, without addition of any conventional fixative) has been demonstrated to produce intracellular precipitates of the insoluble salts of calcium, magnesium, and sodium and to preserve the general cell morphology. In both animal and plant tissues, the electron-opaque antimonate precipitates were found deposited in the nucleus—as well as within the nucleolus—and in the cytoplasm, largely at the site of the ribonucleoprotein particles; the condensed chromatin appeared relatively free of precipitates. The inorganic cations are probably in a loosely bound state since they are not retained by conventional fixatives. The implications of this inorganic cation distribution in the intact cell are discussed in connection with their anionic counterparts, i.e., complexing of cations by fixed anionic charges and the coexistence of a large pool of inorganic orthophosphate anions in the nucleus and nucleolus.     

An improved method for the subcellular localization of calcium using a modification of the antimonate precipitation technique

A new variation of the antimonate precipitation technique, employing tannic acid in the primary aldehyde-antimonate fixative, is described for use in the subcellular localization of calcium in various tissues. Chelation studies and electron microscopic, X-ray microanalytical studies of antimonate precipitates in etiolated oat tissues indicate that calcium is the major cation localized using the present experimental protocol. Preservation of ultrastructural morphology in these tissues is greatly improved over that observed in tissues fixed with conventional antimonate-aldehyde or antimonate-osmium fixatives. The regularity and reproducibility of tissue precipitate patterns suggests that 1) penetration of the tissue by the fixative, and subsequent precipitation of calcium, is rapid and uniform and 2) ion displacement during sample preparation is negligible. Calcium appears to be immobilized efficiently in situ, with greater than 90% 45Ca retention in radiolabeled tissues prepared for electron microscopy. Quantitative aspects of calcium precipitation by antimonate in 45Ca-labeled CaCl2 solutions were examined over a wide range of calcium concentrations. Precipitation was essentially linear over the expected range of biological concentrations of calcium. Furthermore, the 3:1 antimonate to calcium ratio estimated for test tube precipitates was also established for Sb/Ca in tissue precipitates analyzed using energy dispersive x-ray microanalytical (EDX) techniques. These observations suggest that the present technique is potentially useful in the semiquantitative estimation of tissue calcium levels.     

Microgravity and clinorotation cause redistribution of free calcium in sweet clover columella cells

In higher plants, calcium redistribution is believed to be crucial for the root to respond to a change in the direction of the gravity vector. To test the effects of clinorotation and microgravity on calcium localization in higher plant roots, sweet clover (Melilotus alba L.) seedlings were germinated and grown for two days on a slow rotating clinostat or in microgravity on the US Space Shuttle flight STS-60. Subsequently, the tissue was treated with a fixative containing antimonate (a calcium precipitating agent) during clinorotation or in microgravity and processed for electron microscopy. In root columella cells of clinorotated plants, antimonate precipitates were localized adjacent to the cell wall in a unilateral manner. Columella cells exposed to microgravity were characterized by precipitates mostly located adjacent to the proximal and lateral cell wall. In all treatments some punctate precipitates were associated with vacuoles, amyloplasts, mitochondria, and euchromatin of the nucleus. A quantitative study revealed a decreased number of precipitates associated with the nucleus and the amyloplasts in columella cells exposed to microgravity as compared to ground controls. These data suggest that roots perceive a change in the gravitational field, as produced by clinorotation or space flights, and respond respectively differently by a redistribution of free calcium.     

Selective subcellular localization of cations with variants of the potassium (pyro)antimonate technique.

Fixation of rat parotid with an unbuffered osmium tetroxide solution containing nearly saturated potassium (pyro)antimonate resulted in abundant deposition of cation-antimonate precipatates in acinar cells. Altering the antimonate concentration, including buffers or chelators in the solution or changing the primary fixative resulted in an altered intensity and distribution of the precipitates formed in the tissue, apparently reflecting a degree of selectivity in ion localization. Decreasing the concentration of pyroantimonate to about half-saturation preserved predominantly the less soluble antimonate salts (e.g., Na+, Ca++) and resulted in preferential retention of deposits along the plasmalemma and in mitochondrial "dense bodies," with loss of most cytoplasmic and nuclear precipitates. A similar pattern was seen if fixation with the high concentration antimonate-osmium procedure was followed by a prolonged rinse. Adding phosphate or collidine buffers markedly decreased precipitates in the nuclei and on granular reticulum as well. Phosphate buffer or ehtyleneglycoltetraacetate inhibited in vitro precipitation of calcium and sodium and decreased or abolished plasmalemmal deposits. Glutaraldehyde fixation, either in the presence of antimonate or prior to antimonate-containing osmium tetroxide, abolished heterochromatin deposits. Mitochondrial dense bodies were of two types, one containing precipitate and the other inherently osmiophilic. The latter were also observed in pyrophosphate-osmium controls. Results from in vitro titrations of cations with the various antimonate methods and from neutron activation analyses of fixed tissues supported conclusions drawn from fine structural distribution patterns and were interpreted as follows. In rat parotid acinar cells, deposits in heterochromatin and on granular reticulum probably arose from precipitation in sites of high K+ and H+ as well as--NH3+-rich histones. Plasmalemmal antimonate deposits demonstrated sites of sodium and/or calcium accumulation. Some mitochondrial dense bodies contained Ca++ whereas others were inherently osmiophilic. Large, extracellular deposits were probably predominantly sodium precipitates.     

Sodium and calcium localization in cells and tissues by precipitation with antimonate: a quantitative study

Komnick's antimonate technique, which was devised to localize Na+ in cells and tissues, was studied quantitatively. Some modifications, as well as its application to Ca2+ localization, were also investigated. We combined measurements of Na+ and Ca2+ retention in plant roots during the various procedures, electron microscopy, autoradiography, and semiquantitative X-ray microanalysis. We were able to show that (at least in barley roots) antimonate does not precipitate at all with Na+, irrespective of the Na+ content of the tissue or the method of antimonate application. (Even during precipitative freeze dissolution or after freeze drying, no Na+ is precipitated.) By means of Komnick's antimonate technique Ca2+ is trapped within the tissue, but only after serious dislocation. Perspectives for reliable localization of diffusible ions in cells and tissues, by precipitation simultaneously with conventional fixations, are bad.     

水稻非花粉型细胞质雄性不育系及其保持系花药发育过程中Ca2+的分布变化

钙在高等植物中被称为第二信使,与植物的有性生殖有关。为了研究水稻（Oryza sativa L.）花药中钙的定位与花粉败育的关系,利用焦锑酸钾沉淀法研究了非花粉型细胞质雄性不育系G37A及其保持系G37B花药的发育过程及其细胞中Ca^2＋ 的分布变化。研究发现,在2个材料间花药中钙的分布存在大量差异。G37B的可育花药在花粉母细胞时期及二分体时期,很少看到有Ca^2＋的沉积;而在单核花粉时期,Ca^2＋沉积急速地增加,主要定位在绒毡层细胞、花粉外壁外层及乌氏体的表面;随后花药壁上沉积的Ca^2＋减少而花粉的外壁外层仍然有很多Ca^2＋沉积物。相反,G37A的不育花药在花粉母细胞时期和二分体时期有大量的Ca^2＋沉积在小孢子母细胞和花药壁,中间层和绒毡层特别多。在二分体时期之后,不育花药的Ca^2＋沉积减少,特别是绒毡层内切向质膜附近的Ca^2＋几乎消失。但是同时期的可育花药中,有大量的Ca^2＋沉积在绒毡层。不育花药的Ca^2＋沉积在开花几天后消失。根据研究结果推测在不育花药发育早期中更多的钙离子与花粉败育有一定的关系。     

Calcium in the synergid cells and other regions of pearl millet ovaries

Summary The synergids and other cells of mature, unpollinated pearl millet ovaries were investigated using: (1) freeze-substitution fixation in conjunction with scanning electron microscope observations and energy-dispersive X-ray microanalysis to localize total calcium (Ca) and other elements, and (2) antimonate precipitation to selectively localize loosely sequestered, exchangeable calcium (Ca⁺⁺). In freeze-fixed ovaries, the synergid cells, ovary wall, nucellus, and other regions of the ovary displayed, respectively and relatively, extremely high, high, moderate, and low levels of Ca. In antimonate-fixed ovaries, Ca-containing antimonate precipitates exhibited similar distribution patterns. In ovaries fixed using the conventional 2% (w/v) antimonate in fixatives, the synergids were disrupted due to precipitate overload. In the ovary wall, precipitates were mainly located in the intercellular spaces. Some precipitates were observed at the micropyle and along the outer ovule integument, associated with diffuse extracellular material, and in the cell walls of nucellar cells proximal to the micropyle. Examination of precipitate distribution inside the synergids was possible in ovaries fixed using 0.5% (w/v) antimonate in the fixatives. Cytoplasmic organelles of all synergids examined exhibited variable states of disintegration. The amount of precipitates associated with the degenerated organelles appeared to be proportional to the degree of their degeneration. Distinct precipitates were localized in contiguous regions of the nucellar cells fused with the embryo sac, the micropylar half of the embryo sac wall, and the filiform apparatus. The results are discussed in relation to the involvement of Ca⁺⁺ in mediating the functions of synergid cells during fertilization in angiosperms.On Specific Cooperative Agreement 58-43YK-8-0026 with the Department of Biochemistry, University of Georgia, Athens, GA 30602, USA     

Sodium and calcium localization in cells and tissues by precipitation with antimonate: A quantitative study

Summary Komnick's antimonate technique, which was devised to localize Na⁺ in cells and tissues, was studied quantitatively. Some modifications, as well as its application to Ca²⁺ localization, were also investigated.We combined measurements of Na⁺ and Ca²⁺ retention in plant roots during the various procedures, electron microscopy, autoradiography, and semiquantitative X-ray microanalysis. We were able to show that (at least in barley roots) antimonate does not precipitate at all with Na⁺, irrespective of the Na⁺ content of the tissue or the method of antimonate application. (Even during precipitative freeze dissolution or after freeze drying, no Na⁺ is precipitated.) By means of Komnick's antimonate technique Ca²⁺ is trapped within the tissue, but only after serious dislocation. Perspectives for reliable localization of diffusible ions in cells and tissues, by precipitation simultaneously with conventional fixations, are bad.     

Alterations in ultrastructure and subcellular localization of Ca2+ in poplar apical bud cells during the induction of dormancy

In poplar (Populus deltoides Bartr. ex Marsh), bud dormancyand freezing tolerance were concomitantly induced by short-day(SD) photoperiods. Ultrastructural changes and the alterationin subcellular localization of calcium in apical bud cells associatedwith dormancy development were investigated. During the developmentof dormancy, the thickness of cell walls increased significantly,the number of starch granules increased, and there was a significantaccumulation of storage proteins in the vacuoles of the apicalbud cells. The most striking change was the constriction andblockage of the plasmodesmata. It was demonstrated that antimonate precipitation is a reliabletechnique for studying subcellular localization of calcium inpoplar apical bud cells. Under the long day (LD) photoperiod,electron-dense calcium antimonate precipitates were mainly localizedin vacuoles, intercellular spaces and plastids. Some antimonateprecipitates were also found in the cell walls and at the entranceof the plasmodesmata. However, there were few Ca²⁺ depositsfound in the cytosol and nucleus. After 20 d of SD exposure,when development of bud dormancy was initiated, calcium depositsin intercellular spaces were decreased, whereas some depositswere found in the cytosol and nuclei. From 28–49 d ofSD exposure, while dormancy was developing, a large number ofCa²⁺ precipitates were found in the cytosol and nuclei. Whendeep dormancy was reached after 77 d of SD exposure, Ca²⁺ depositsbecame fewer in both cytosol and nuclei, whereas numerous depositswere again observed in the cell walls and in the intercellularspaces. These results suggest that under the influence of SDphotoperiods, there are alterations in subcellular Ca²⁺ localization,and changes in ultrastructure of apical bud cells during thedevelopment of dormancy. The constriction and blockage of plasmodesmatamay cause the cessation of symplastic transport, limit cellularcommunication and signal transduction between adjacent cells,which in turn may lead to events associated with growth cessationand dormancy development in buds. Key words: Poplar, apical bud cells, Ca²⁺ subcellular localization, dormancy     

Localization of calcium in the pericarp cells of tomato fruits during the development of blossom-end rot

Summary. Blossom-end rot (BER) of tomato (Lycopersicon esculentum) fruits is considered to be a physiological disorder caused by calcium deficiency. We attempted to clarify the localization of calcium in the pericarp cells and the ultrastructural changes during the development of BER. Calcium precipitates were observed as electron-dense deposits by an antimonate precipitation method. Some calcium precipitates were localized in the cytosol, nucleus, plastids, and vacuoles at an early developmental stage of normal fruits. Calcium precipitates were increased markedly on the plasma membrane during the rapid-fruit-growth stage compared with their level at the early stage. Cell collapse occurred in the water-soaked region at the rapid-fruit-growth stage in BER fruits. There were no visible calcium precipitates on the traces of plasma membrane near the cell wall of the collapsed cells. The amount of calcium precipitates on plasma membranes near collapsed cells was smaller than that in the cells of normal fruits and normal parts of BER fruits, and the amount on cells near collapsed cells was small. The amount of calcium precipitates on the plasma membranes increased as the distance from collapsed cells increased. On the other hand, calcium precipitates were visible normally in the cytosol, organelles, and vacuoles and even traces of them in collapsed cells. The distribution pattern of the calcium precipitates on the plasma membrane was thus considerably different between normal and BER fruits. On the basis of these observations, we concluded that calcium deficiency in plasma membranes caused cell collapses in BER tomato fruits.Correspondence and reprints: National Institute of Vegetable and Tea Science, National Agricultural Research Organization, Ano, Mie, 514-2392, Japan.     

Localization of Ca++-containing antimonate precipitates during mitosis

Intracellular bound Ca++ has been localized throughout mitosis and cytokinesis in two plant species by means of in situ precipitation with potassium antimonate and electron microscope visualization. Identification of Ca++ as the major cation precipitated was made by comparing solubility properties in water, EDTA, and EGTA of the intracellular deposits with respect to those of K+-, Mg++-, and Ca++- antimonate standards. In spermatogenous cells of the water fern, Marsilea vestita, and stomatal complex cells of barley, Hordeum vulgare, antimonate deposits have been found associated with the endoplasmic reticulum (ER), vacuoles, euchromatin/nucleoplasm, and mitochondria. The last contain a much higher density of precipitates in Marsilea than in Hordeum. Dictyosomes and the nuclear envelope of Marsilea also contain antimonate deposits, as do the plasmalemma, cell wall, and phragmoplast vesicles of Hordeum. Microtubule-organizing centers such as kinetochores and the blepharoplast of Marsilea do not stain. In spite of differences in associated antimonate between certain organelles of the two species, the presence of antimonate aong the ER throughout the cell cycle is common to both. Of particular interest are those precipitates seen along the tubules and cisternae of the extensive smooth ER that surrounds and invades the mitotic spindle in both species. The ability to bind divalent cations makes the mitotic apparatus (MA)-associated ER a likely candidate for regulation of free Ca++ levels in the immediate vicinity of structural components and processes that are Ca++-sensitive and proposed to be Ca++-regulated.     

Calcium distribution in fertilized and unfertilized ovules and embryo sacs of Nicotiana tabacum L.

Potassium antimonate was used to localize Ca²⁺ in tobacco ovules from 0 to 7 d after anthesis in pollinated and emasculated flowers. Antimonate binds “loosely bound” Ca²⁺ into calcium antimonate; less-soluble forms are unavailable and free calcium usually escapes. Ovules are immature at anthesis. Abundant calcium precipitates in nucellar cells surrounding the micropylar canal. A difference between calcium in the two synergids emerges at 1 d, which is enhanced in pollinated flowers. The future receptive synergid accumulates more precipitates in the nucleus, cytoplasm and cell walls. After fertilization, micropyle precipitates diminish, and the ovule is unreceptive to further tube entry. In emasculated flowers 6 d after anthesis, ovular precipitates essentially disappear; however, flowers pollinated at 4–5 d and collected 2 d later largely restore their prior concentration of precipitates. Ovular precipitates occur initially in the nucellus, then the embryo sac, and finally the synergid and micropylar filiform apparatus. Possibility, calcium is released from the embryo sac, although no structural evidence of exudate formation was observed. Calcium precipitates in the ovule correlate with the ability of the ovule to be fertilized, suggesting that successful pollen tube entry and later development may require calcium of the class precipitated by antimonate. Received: 14 August 1996 / Accepted: 9 October 1996     

Calcium distribution in fertile and sterile anthers of a photoperiod-sensitive genic male-sterile rice

Potassium antimonate was used to locate Ca²⁺ in fertile and sterile anthers of a photoperiod-sensitive genic male-sterile rice (Oryza sativa L. japonica). During the development of fertile anthers, abundant calcium precipitates accumulated in the anther walls and on the surface of pollen grains and Ubish bodies at the late developmental stage of the microspore, but not in the cytoplasm of pollen grains. Following the accumulation of starch grains in pollen, calcium precipitates on pollen walls diminished and increased in parenchymatous cells of the connective tissue. In sterile anthers, calcium precipitates were abundant in the middle layer and endothecium, but not in the tapetum, as was found in fertile anthers. A special cell wall was observed between the tapetum and middle layer of sterile anthers that appeared to relate to distinctive calcium accumulation patterns and poor pollen wall formation in the loculi. The formation of different patterns of antimonate-induced calcium precipitates in the anthers of photoperiod-sensitive genic male-sterile rice indicates that anomalies in the distribution of calcium accumulation correlate with the failure of pollen development and pollen abortion. Received: 30 May 1997 / Accepted: 5 July 1997     

The release of membrane-associated calcium from rabbit neutrophils by fixatives. Implications for the use of antimonate staining to localize calcium

Summary The addition of oxalate to a suspension of rabbit peritoneal neutrophils before fixation with glutaraldehyde and postfixation with osmium tetroxide-antimonate greatly enhanced the amount of calcium antimonate precipitate subsequently detectable with the electron microscope. Using chlortetracycline as a fluorescent probe for membrane-associated calcium, it was found that both glutaraldehyde and osmium tetroxide release calcium from membrane-associated stores in suspensions of living neutrophils. These findings suggest that some of the calcium released from cellular stores during fixation with glutaraldehyde is trapped within the neutrophil by oxalate which then reacts with potassium antimonate. This produces a more copious precipitate of calcium antimonate than fixation without oxalate. It is suggested, therefore, that the histochemical localization of calcium by antimonate techniques may not always represent thein vivo situation. The use of oxalate during fixation, however, may give a better indication of the amount of calcium stored within a cell.     

Ultrastructural localization of calcium in post-mortem bovine muscle: A cytochemical and X-ray microanalytical study

Summary Calcium localization was demonstrated in bovine longissimus muscle using the antimonate precipitation technique in combination with electron probe X-ray microanalysis. Samples were taken each hour during the first 24 h post-mortem, and then after a storage period of 8 and 15 days. For all sampling times analysed, heavy precipitates were seen in dense parts of nuclei and on N-lines of myofibrils. Up to 18–20 h post-mortem, deposits were observed in sarcoplasmic reticulum at the level of triads. In comparison with the earlier post-mortem samples, myoplasmic precipitates were strongly increased at 4 h post-mortem, and just before rigor onset, at 19 h where intermyofibrillar spaces were completely blackened and triads were no more visible. These localizations of precipitates were still observed up to 15 days post-mortem. At these storage times, myofibril disruptions were seen at the level of N-lines. Wavelength-dispersive and energy-dispersive spectrometric analyses indicated that significant amounts of calcium occurred in the dense precipitates observed.     

Localization of calcium in roots and microsomal membranes of corn by direct pyroantimonate precipitation

Many cell membrane systems, including microsomal vesicles of corn, are able to regulate calcium levels both in vivo and in vitro, often in an ATP-dependent, calmodulin-stimulated fashion. The purpose of this study was to determine calcium distribution in meristematic cells of intact tissue and microsomal vesicles from corn roots using direct pyroantimonate-osmium fixation. In root cells, precipitates were localized in mitochondria, plastids, the nucleus, endoplasmic reticulum, Golgi apparatus, and along the plasma membrane. Plasma membrane-enriched microsomal vesicles isolated from corn roots incubated in media to permit calcium transport before pyroantimonate-osmium fixation show internal precipitates associated with the membrane and in the lumen of the vesicles. De-staining of the sections with 1 mM EDTA or EGTA removed precipitate from the sections, confirming the presence of calcium in the antimonate precipitates. These data support biochemical data that this same membrane preparation exhibited ATP-dependent calcium sequestration that was stimulated by calmodulin, as measured by retention of 45Ca. This provides evidence that these membranes are responsible for ATP-requiring, calmodulin-stimulated calcium transport in the intact cell.     

Ionic components of secretory cell surfaces in relation to secretory function

Synopsis Rat pancreas was examined by ultrastructural cytochemical methods for localizing cations and anions, as well as polyanions and, more specifically, sulphated mucosubstance. Exceptionally abundant antimonate-precipitable cation was demonstrated between pancreatic acinar cells and at the base of the centro-acinar and other duct epithelial cells. Precipitates of nuclear heterochromatin appeared lighter whereas those of mitochondria and cytoplasm were coarser and more conspicuous in acinar that duct cells. Stimulation with synthetic secretin at a low level diminished antimonate reactivity of nuclei as well as the precipitation at the basement membrane of centro-acinar cells. At a higher dose, secretin selectively eliminated precipitation between and below centro-acinar and other duct cells while inducing increased antimonate-reactive cation in centro-acinar cells and the acinar lumens. Pancreozymin stimulation elimated antimonate-precipitable cations between acinar cells and, to a much lesser extent, those between duct cells and increased cytoplasmic precipitates on granular reticulum of acinar cells.Silver-precipitable anions were localized on the luminal surface of the apical plasma lemma and the outer surface of the latero-basal plasmalemma of centro-acinar cells but not on acinar cell surfaces. Silver precipitates also occurred on junctional complexes of acinar and duct epithelial cells and at tight junctions of acinar cells and on the inner face of the lateral plasmalemma of acinar cells.Dialysed iron staining demonstrated the most number of sites of acid mucosubstance on the luminal surface of the plasmalemma of acinar cells. Lateral and basal plasmalemmas of centro-acinar and more distal duct cells stained lightly with dialysed iron but those of acinar cells did not. Dialysed iron visualized acid mucosubstance in the lamina lucida of the basement membrane of duct but not of acinar cells. Dialysed iron staining of the plasma membranes succumbed to prior sialidase treatment whereas that of basement membrane resisted digestion. High iron diamine staining demonstrated sulphated mucosubstance in the lamina lucida of the duct basement membrane exclusively. The cytochemical results implicate centro-acinar cells as primarily responsible for contributing fluid and electrolytes to pancreatic secretion.