An ultrastructural double-labelling method: immunohistochemical localization of cell adhesion molecule L1 on HRP-labelled developing corticospinal tract axons in the rat

A double electronmicroscopical (EM) staining was developed which enabled the ultrastructural localization of cell adhesion molecules on the outer axonal membrane of horseradish peroxidase (HRP)-labelled axons in the developing central nervous system (CNS). HRP was used to anterogradely trace outgrowing corticospinal tract (CST) axons in ten-day-old rats. After visualization of HRP using tetramethylbenzidine (TMB) as a chromogen and ammoniumheptamolybdate (AHM) as a stabilizer at pH 6.0 as described previously (Joosten et al. 1987, J Histochem Cytochem 35: 623-626) an additional diaminobenzindine (DAB)-Ni incubation was carried out for further stabilization. Subsequently a preembedding immunoperoxidase (DAB) staining was executed for detection of cell adhesion molecule L1. Using this procedure anterogradely HRP-labelled CST axons were recognizable by a granular black TMB-AHM-DABNi reaction product at the light microscopic (LM) level, which clearly contrasts to the relatively homogeneous brown L1-immunostaining. Electronmicroscopically HRP-labelled CST axons were characterized by the presence of an intracellular crystaloid TMB-AHM-DABNi reaction product which made identification of CST axons rather easy, whereas the L1-DAB precipitate could be noted on the outer axonal membrane of the HRP-labelled CST axons, marking the presence of the L1 cell adhesion molecule. In addition the procedure described in this report preserves ultrastructural details of developing neural tissue. In conclusion, the method presented can be employed in combined HRP-tracing and immunohistochemical electronmicroscopic studies.     

An ultrastructural double-labelling method: immunohistochemical localization of cell adhesion molecule L1 on HRP-labelled developing corticospinal tract axons in the rat

Summary A double electronmicroscopical (EM) staining was developed which enabled the ultrastructural localization of cell adhesion molecules on the outer axonal membrane of horseradish peroxidase (HRP)-labelled axons in the developing central nervous system (CNS). HRP was used to anterogradely trace outgrowing corticospinal tract (CST) axons in ten-day-old rats. After visualization of HRP using tetramethylbenzidine (TMB) as a chromogen and ammoniumheptamolybdate (AHM) as a stabilizer at pH 6.0 as described previously (Joosten et al. 1987, J Histochem Cytochem 35:623–626) an additional diaminobenzindine (DAB)-Ni incubation was carried out for further stabilization. Subsequently a pre-embedding immunoperoxidase (DAB) staining was executed for detection of cell adhesion molecule L1. Using this procedure anterogradely HRP-labelled CST axons were recognizable by a granular black TMB-AHM-DABNi reaction product at the light microscopic (LM) level, which clearly contrasts to the relatively homogeneous brown L1-immunostaining. Electronmicroscopically HRP-labelled CST axons were characterized by the presence of an intracellular crystaloid TMB-AHM-DABNi reaction product which made identification of CST axons rather easy, whereas the L1-DAB precipitate could be noted on the outer axonal membrane of the HRP-labelled CST axons, marking the presence of the L1 cell adhesion molecule. In addition the procedure described in this report preserves ultrastructural details of developing neural tissue. In conclusion, the method presented can be employed in combined HRP-tracing and immunohistochemical electronmicroscopic studies.     

Cytochemical visualization of anions in collagenous and elastic fiber-associated connective tissue matrix in neonatal and adult rat lungs using iron-containing stains

The cytochemical reactivity of pulmonary connective tissue matrix component in neonatal and adult rat was evaluated using high iron diamine (HID) to detect sulfate ester end groups and dialyzed iron (DI) to detect sulfated and carboxylated end groups of complex carbohydrates, including glycoproteins and glycosaminoglycans at the ultrastructural level. The HID reaction product, in the form of discrete 5-12 nm silver particles following appropriate intensification with thiocarbohydrazide-silver proteinate, was found associated with cell surfaces, the elastin component of elastic fibers, and at regular intervals along the length of collagen fibers in large airways and deep lung interstitium. Staining was similar in adult and neonatal rats, except in areas where connective tissues were presumably still rapidly developing in the neonatal animals. Here large gaps or spaces containing filamentous structures were observed between collagen and elastic fibers. The distribution of DI-reactive sites was similar to that seen with HID with the exception of elastic fibers in which only the microfibrillar portion stained. The collagen-associated reaction was not regularly disposed like that stained with HID, but rather it formed a tight continuous density around the fiber. These results indicated the presence and location of glycoproteins and glycosaminoglycans in connective tissue ground substance regions prior to the full development of elastic and collagenous elements in neonatal pulmonary airways and parenchyma. They also demonstrate cytochemically the presence of a sulfate ester-containing complex sugar found associated with the elastin component of elastic fibers in the lung.     

Cytochemical visualization of anions in collagenous and elastic fiber-associated connective tissue matrix in neonatal and adult rat lungs using iron-containing stains

Summary The cytochemical reactivity of pulmonary connective tissue matrix components in neonatal and adult rat was evaluated using high iron diamine (HID) to detect sulfate ester end groups and dialyzed iron (DI) to detect sulfated and carboxylated end groups of complex carbohydrates, including glycoproteins and glycosaminoglycans at the ultrastructural level. The HID reaction product, in the form of discrete 5–12 nm silver particles following appropriate intensification with thiocarbohydrazide-silver proteinate, was found associated with cell surfaces, the elastin component of elastic fibers, and at regular intervals along the length of collagen fibers in large airways and deep lung interstitium. Staining was similar in adult and neonatal rats, except in areas where connective tissues were presumably still rapidly developing in the neonatal animals. Here large gaps or spaces cointaining reactive filamentous structures were observed between collagen and elastic fibers. The distribution of DI-reactive sites was similar to that seen with HID with the exception of elastic fibers in which only the microfibrillar portion stained. The collagen-associated reaction was not regularly disposed like that stained with HID, but rather it formed a tight continuous density around the fiber. These results indicated the presence and location of glycoproteins and glycosaminoglycans in connective tissue ground substance regions prior to the full development of elastic and collagenous elements in neonatal pulmonary airways and parenchyma. They also demonstrate cytochemically the presence of a sulfate ester-containing complex sugar found associated with the elastin component of elastic fibers in the lung.Supported by Public Health Servece Grant HL 24748     

Electron microscopic immunocytochemical investigation on the postnatal development of the vasopressin system in the rat

Summary The present ultrastructural results indicate that, in the rat, the vasopressin-synthesizing perikarya of the supraoptic nucleus (NSO) attain a certain degree of maturity earlier than those of the paraventricular nucleus (NPV). In the neonate rat, the stainability of the nuclear areas is very weak; in the perikarya of the NSO a few labeled granules can be found, whereas the perikarya of the NPV often display only a labeled Golgi area, the cytoplasm being devoid of granules. At the end of the first (NSO) and the second (NPV) postnatal weeks, the filling of the neurosecretory granules with vasopressin is inhomogeneous with irregular spots of reaction product distributed on the granules. This feature is less obvious during the following week and has nearly disappeared after the third and fourth postnatal weeks. Already in the neonate two types of vasopressin-positive fibers are observed in the median eminence, characterized by the different diameters of their granules and by their typical location in the internal and the external pericapillary contact zone. Especially in one and two week-old animals, in the internal zone of the median eminence and, to a lesser degree in the neural lobe, the immunocytochemical reaction product is deposited on an axonal tubular network. Judging from the presence of very few vasopressin-negative fibers in the neural lobe of the neonate, the development of the oxytocin system appears to be delayed. A characteristic relationship between pituicytes and the neurosecretory fibers can be observed during the first two postnatal weeks. After the third postnatal week the immunocytochemical features of the vasopressin system correspond approximately to that in adult rats.Supported by the Deutsche Forschungsgemeinschaft (Grant Nr. Kr 569/3) and Stiftung Volkswagenwerk     

Ipsilateral,ventral corticospinal tract of the adult rat: Ultrastructure,myelination and synaptic connections

The corticospinal tract (CST) of the rat is a widely used model system in developmental, physiological, and regeneration studies. The CST of the rat consists of a main tract, that runs in the dorsomedial funiculus and several minor components. We have shown earlier that one of the minor components, the ipsilateral, ventral CST, projects all the way down the spinal cord in the adult rat and single fibers form large terminal arbors in their spinal target areas. Here we investigated its ultrastructure and compared it to that of CST fibers of the main tract. By the use of anterograde axonal tracing with biotin dextran-amine (BDA) and pre-embedding avidin-peroxidase histochemistry we investigated axon diameters and myelination using electron microscopy. Ipsilateral, ventral CST fibers were found to run in the ventral funiculus close to the midline. They were intermingled with heavily myelinated large diameter axons, presumably reticulospinal, vestibulospinal, or tectospinal fibers. Ipsilateral, ventral CST fibers were of small diameter (0.68 μm, ±0.04) and about [frac34] of them were moderately myelinated (9.64 ± 0.7 layers of myelin). Co-localization of a rhodamine-dextrane anterograde tracer with the presynaptic marker synaptophysin using confocal microscopy and electron microscopy revealed varicosities on terminal arborisations to be presynaptic boutons and clearly demonstrated contacts to neurons in intermediate laminae of the spinal cord at lumbar spinal levels. This study extends our earlier work indicating that the ipsilateral, ventral CST component of the adult rat is a morphologically complete CST component and may perform similar functions to the main CST component.     

The use of unfixed cryostat sections for electron microscopic study of D-amino acid oxidase activity in rat liver.

Unfixed cryostat sections of rat liver were incubated to demonstrate D-amino acid oxidase activity at the ultrastructural level. Incubation was performed by mounting the sections on a semipermeable membrane which was stretched over a gelled incubation medium containing D-proline as substrate and cerium ions as capture reagent for hydrogen peroxide. After an incubation period of 30 min, ultrastructural morphology was retained to such an extent that the final reaction product could be localized in peroxisomes, whereas the crystalline core remained unstained. Control incubations were performed in the absence of substrate; the lack of final reaction product in peroxisomes indicated the specificity of the reaction. We conclude that the semipermeable membrane technique opens new perspectives for localization of enzyme activities at the ultrastructural level without prior tissue fixation, thus enabling localization of the activity of soluble and/or labile enzymes.     

Ultrastructural immunolocalization of lysyl oxidase in vascular connective tissue

The localization of lysyl oxidase was examined in calf and rat aortic connective tissue at the ultrastructural level using polyclonal chicken anti-lysyl oxidase and gold conjugated rabbit anti-chicken immunoglobulin G to identify immunoreactive sites. Electron microscopy of calf aortic specimens revealed discrete gold deposits at the interface between extracellular bundles of amorphous elastin and the microfibrils circumferentially surrounding these bundles. The antibody did not react with microfibrils which were distant from the interface with elastin. There was negligible deposition of gold within the bundles of amorphous elastin and those few deposits seen at these sites appeared to be associated with strands of microfibrils. Lysyl oxidase was similarly localized in newborn rat aorta at the interface between microfibrils and nascent elastin fibers. Gold deposits were not seen in association with extracellular collagen fibers even after collagen-associated proteoglycans had been degraded by chondroitinase ABC. However, the antibody did recognize collagen-bound lysyl oxidase in collagen fibers prepared from purified collagen to which the enzyme had been added in vitro. No reaction product was seen if the anti-lysyl oxidase was preadsorbed with purified lysyl oxidase illustrating the specificity of the antibody probe. The present results are consistent with a model of elastogenesis predicting the radial growth of the elastin fiber by the deposition and crosslinking of tropoelastin units at the fiber-microfibril interface.     

Light- and electron microscopic localization of vasopressin or a vasopressin-like substance in the neurons of the rat suprachiasmatic nucleus

Summary In the suprachiasmatic nucleus of the rat light microscopic immunostaining for vasopressin reveals a distribution pattern of the immunoreactive material different from that known for the supraoptic nucleus. Among non-stained neurons positive-reacting perikarya display a cap- or tiplike labeling. The area of the suprachiasmatic nucleus is marked by delicate vasopressin-positive fibers. At the ultrastructural level the reaction product, after incubation with anti-vasopressin, is localized in small elementary granules unevenly distributed over the cytoplasm. Groups of axons containing specifically labeled granules contact non-reacting fibers.Supported by the Deutsche Forschungsgemeinschaft (Grant Nr. Kr. 569/2) and Stiftung Volkswagenwerk     

1-naphthol-pyronin B as a novel substrate for silver intensification: application to light and electron microscopic immunocytochemistry of neuroendocrine systems

We describe a modification of silver intensification of immunoperoxidase end-product using 1-naphthol (1N) and 1N enhanced by pyronin B after suppressing nonspecific tissue argyrophilia with a solution of penicillamine and merthiolate buffered near neutral pH. This approach facilitates the preservation of a second antigen sequentially labeled in the same tissue section for light microscopic double immunolabeling experiments and also allows retention of ultrastructural detail. Using this protocol, we obtained rapid and uniform silver intensification of somatostatin (SRIF)-immunoreactive (IR) neuronal perikarya and processes in the rat hypothalamic paraventricular nucleus (PVN). Ultrastructurally, 1N- and 1N-pyronin B-silver intensified reaction product was clearly recognized by the presence of a coarse intracellular precipitate of high electron density. Light microscopic double-immunolabeling studies demonstrated the association between SRIF- and thyrotropin-releasing hormone (TRH)-IR neuronal systems in the PVN. We propose that silver intensification of 1N and 1N-pyronin B is a useful alternative to standard methods of silver intensification of immunoperoxidase reaction product at both light and ultrastructural levels and may be particularly amenable for double-immunolabeling studies.     

CD56(NCAM) antigen in glandular epithelium of human thyroid: light microscopic and ultrastructural study

CD56 antigen, an isoform of the neural cell adhesion molecule (NCAM) was previously found by us in human thyroid by APAAP immunohistochemistry in light microscopy on frozen tissue sections. In the current study, it was attempted to trace the antigen in question using another light microscopic immunohistochemical procedure and to validate the results at the ultrastructural level. For light microscopy, cryostat sections of 12 surgical samples of human thyroid were subjected to ABC (preformed avidin-biotin-peroxidase complex) method. For immunoelectron microscopy, immunoperoxidase reaction was carried out on prefixed, small thyroid tissue blocks. Following preliminary inspection of semithin sections, ultrathin sections were examined in the transmission electron microscope. ABC reaction revealed distinct specific CD56 staining of thyrocyte cell membranes. The staining was weak or absent in thyroid papillary carcinoma cells. The results were confirmed in semithin sections by indirect immunoperoxidase. The latter reaction in ultrathin sections at the ultrastructural level has shown that specific reaction product was confined to free and lateral surfaces of thyroid follicular cells. Endothelial cell membranes of thyroid capillary vessels were totally devoid of the reaction product. The reaction was weakly positive in thyroid follicular and papilllary carcinomas but absent from medullary carcinoma.     

Morphological and physiological regeneration in the auditory system of adult <Emphasis Type="Italic">Mecopoda elongata</Emphasis> (Orthoptera: Tettigoniidae)

Orthopterans are suitable model organisms for investigations of regeneration mechanisms in the auditory system. Regeneration has been described in the auditory systems of locusts (Caelifera) and of crickets (Ensifera). In this study, we comparatively investigate the neural regeneration in the auditory system in the bush cricket Mecopoda elongata. A crushing of the tympanal nerve in the foreleg of M. elongata results in a loss of auditory information transfer. Physiological recordings of the tympanal nerve suggest outgrowing fibers 5 days after crushing. An anatomical regeneration of the fibers within the central nervous system starts 10 days after crushing. The neuronal projection reaches the target area at day 20. Threshold values to low frequency airborne sound remain high after crushing, indicating a lower regeneration capability of this group of fibers. However, within the central target area the low frequency areas are also innervated. Recordings of auditory interneurons show that the regenerating fibers form new functional connections starting at day 20 after crushing.     

Ultrastructure of the vocal muscle and its morphological differences from the myocardium]

A comparative ultrastructural investigation of the M. vocalis in mammals has been carried out. Morphological differences between the vocal muscle and cardiac tissue are reported; a distinct classification of the M. vocalis according to a typisation of skeletal muscle fibers is presented. In all species investigated (man, dog, cat, guinea-pig and rat) the general ultrastructure of the sarcomeres as well as their mitochondrial content and the innervation pattern allow to classify the M. vocalis as to belong to the "fast twitch (white) skeletal muscle fibers. A single innervation was found with large motor endplates containing numerous synaptic infoldings. Structural specializations known to be characteristic for cardiac tissue, e.g. intercalated discs, T-tubules at the level of the Z-band and nuclei in a midst postion of the muscle cell could not be observed. The m. vocalis, therefore, cannot be considered to have histologically any relationship with cardiac tissue. The vocal muscle is described as a special type of skeletal muscle very similar to the extraocular muscles. The electron microscopic findings are discussed with respect to current theories of phonation. The myoleastic theory of phonation can be favoured according to our ultrastructural results.     

NUCLEOSIDE PHOSPHATASE AND CHOLINESTERASE ACTIVITIES IN DORSAL ROOT GANGLIA AND PERIPHERAL NERVE

In dorsal root ganglia and peripheral nerve of the rat and other species, nucleoside phosphatase and unspecific cholinesterase reaction products are found in the plasma membranes and spaces between them at two sites: (1) Schwann cell-axon interfaces and mesaxons of unmyelinated fibers, and (2) sheath cell-perikaryon interfaces and interfaces between adjacent sheath cells. Acetylcholinesterase reaction product is found in the perikaryon (within the endoplasmic reticulum) and the axon (axoplasmic surface). Nucleoside phosphatase reaction product is also found in the numerous vacuoles at the surface of perineurium cells, ganglion sheath cells, and cells surrounding some ganglion blood vessels. Nucleoside phosphatase activities in the sections fail to respond, in the manner described for "transport ATPase," to diisopropylphosphofluoridate, sodium and potassium ions, and ouabain. Nucleoside diphosphates are hydrolyzed more slowly than triphosphates in unmyelinated fibers, and are not hydrolyzed at the perikaryon surface. Nucleoside monophosphates are either not hydrolyzed or hydrolyzed very slowly. In contrast to these localizations, which are believed to demonstrate sites of enzyme activity, it is considered likely that diffusion artifacts account for the nucleoside phosphatase reaction product frequently found along the outer surfaces of myelinated fibers and within vacuoles at the Schwann cell surfaces of these fibers. The diffuse reaction product seen in basement membranes of ganglion and nerve may also be artifact.     

Development of the innervation of long bones: expression of the growth-associated protein 43

It has been known from clinical and experimental observations that the peripheral nervous system is involved in the development of long bones. Expression of growth-associated protein 43 (GAP-43/B-50) was found in axonal growth cones during embryonic and postnatal ontogeny as well as in regenerating axons after nerve injury. The aim of the present study was to examine the occurrence of growing nerve fibers in rat tibia from gestational day 16 (GD 16) to postnatal day 28 (PD28). An indirect immunoenzymatic reaction using antibodies raised against GAP-43 was applied to detect outgrowing nerve fibers penetrating into the developing bone. On GD 16 and GD 17 no GAP-43-immunoreactive (IR) fibers were observed in the close vicinity of bone rudiments. On GD19 GAP-43-IR fibers were scarcely present within the periosteum of the central portion of the diaphysis. In the perichondrium surrounding the proximal epiphysis, nerve fibers were first detected around birth. From PD1 onward, numerous fibers were seen in the fibrous buds of the perichondrium at the epi-metaphyseal junction (Ranvier's grooves), some of them being adjacent to the blood vessels. Nerve fibers penetrating into the bone and located in the bone marrow, predominantly associated with blood vessels, were first observed on GD21 and their number increased with further development. They were initially located in the central portion of the diaphysis and later extended towards the metaphyses. On PD4 an increased number of GAP-43-IR fibers appeared in the perichondrium of proximal and distal epiphyses. In the fibrous strands penetrating into the epiphyses and in the secondary ossification centers, nerve fibers were first observed on PD10. From PD14 onward the pattern of tibial innervation remained unchanged but the intensity of GAP-43 immunostaining visibly decreased. The present study demonstrates that developing long bones of rat hindlimbs are supplied by growing nerve fibers immunoreactive for GAP-43 from GD 19 onward. Time and location of their appearance were at least partially correlated with known events taking place during long bone development, e.g. formation of primary and secondary ossification centers. Decreased expression of GAP-43 immunoreactivity in later developmental stages is believed to reflect nerve fiber maturation.     

A light and electron microscopic procedure for sequential double antigen localization using diaminobenzidine and benzidine dihydrochloride

Very few double-antigen staining methods are available that are applicable to both light and electron microscopy. The objective of this study was to develop for localization of two neural antigens simultaneously a procedure which would be sensitive, simple to perform, offer permanent reaction products, and permit correlated light and ultrastructural analysis. The method employs sequential immunoperoxidase staining without antibody elution, in which the first sequence of antibodies is visualized with 3,3'-diaminobenzidine (DAB) and the second with benzidine dihydrochloride (BDHC). The DAB reaction product (brown and diffuse) was easily distinguishable from the BDHC deposit (blue, granular, and more electron-dense) by both light and electron microscopy. The procedure was used to simultaneously localize choline acetyltransferase-and either substance P or tyrosine hydroxylase in rat brain at both light and ultrastructural levels. Control experiments demonstrated the absence of both color mixing and antibody crossreactions, even when both primary antibodies were from the same species. This study demonstrates the usefulness of BDHC as a chromogen for immunoperoxidase staining either alone or in combination with DAB, and describes a double method which should have wide applicability for detailed studies of most pairs of antigens at both light and ultrastructural levels.     

Topography of cerebroside sulfotransferase in Golgi-enriched vesicles from rat brain

Cerebroside sulfotransferase (CST) catalyzes the final step in the synthesis of sulfatide (sulfogalactocerebroside) by transferring the sulfate from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to galactocerebroside. Orientation of CST was studied in vesicles enriched in this enzyme obtained from 21-d-old rat brain. Several lines of evidence indicate that CST is located on the luminal side of these vesicles. (a) Sulfation of endogenous galactocerebroside occurred in vesicles only in the presence of a detergent to render the membranes permeable to exogenous PAPS. (b) There is a pool of latent enzyme within the vesicle, which is released by Triton X-100. (c) CST is not destroyed by trypsin unless the vesicle membranes are first made permeable by Triton X-100. (d) Glycolipid substrate, when covalently attached to agarose beads, was not sulfated unless the enzyme was solubilized. These results are similar to those obtained with thiamine pyrophosphatase, which is known to be located within the lumen of the vesicles. This study establishes that an enzyme synthesizing a complex glycolipid is localized within Golgi-enriched vesicles. Since the product of the CST reaction must also be localized to the luminal side of the vesicles, it is most likely that sulfatide is located at the intraperiod line (outer layer) of myelin. The orientation of CST within the vesicle provides a mechanism for the asymmetrical assembly of glycolipids in bilayers.     

Nerve fiber production by intraocular adrenal medullary grafts: Stimulation by nerve growth factor or sympathetic denervation of the host iris

Summary This study evaluates the production of adrenergic nerve fibers by adrenal medullary tissue of the adult rat grafted to the anterior chamber of the eye of adult recipients. The chromaffin grafts attach to and become vascularized by the host iris. They decrease in size intraocularly during the first 3 weeks. This decrease is somewhat counteracted by sympathetic denervation of the host iris, and better counteracted by sympathetic denervation and addition of nerve growth factor (NGF, given at grafting and 1 and 2 weeks after grafting). Outgrowth of adrenergic nerve fibers from the grafts into the host iris was studied in wholemount preparations by use of the Falck-Hillarp technique 3 weeks after grafting. The innervated area of the host iris was approximately doubled in the chronically sympathectomized group and doubled again in the chronically sympathectomized NGF-supplemented group. Chronic sympathetic denervation had no effect on density of outgrowing nerves, whereas addition of NGF more than doubled nerve density. Since sympathetic denervation causes a slight elevation of NGF activity in the iris, the present experiments are taken as evidence that the level of NGF in the iris regulates formation of nerve fibers by adrenal medullary tissue grafts from adult rats.     

Immunocytochemical localization of S-100 protein in the brain of adult rat

Summary The cellular and subcellular distribution of the nervous system-specific S-100 protein has been investigated in the brain of adult rat at the ultrastructural level by the pre-embedding unlabelled antibody PAP method. The protein is found in both fibrous and protoplasmic astrocytes and in the ependymal cells. The neurons, the oligodendrocytes as well as the microglial cells are lacking S-100. The labelled cells show a reaction product diffusely distributed in the cytoplasmic matrix and on specialized membranes, namely plasma membranes, outer mitochondrial membranes and membranes of the endoplasmic reticulum and Golgi apparatus. The astrocytic filaments and the axonemes of the ependymal cilia exhibit a strong immunoreactivity. The reaction product is also present in the nucleoplasm of the astrocytes and ependymal cells but it is absent from the nucleolus and nuclear envelope. This immunocytochemical data on tissue with satisfactory ultrastructural preservation, provides new information on the localization of the S-100 protein, and could contribute to the understanding of the biological role of the protein.