Pre-embedding Staining Research Articles

Morphological evidence for the association of plasma membrane glycoprotein IIb/IIIa with the membrane skeleton in human platelets

We examined the association between glycoprotein (GP) IIb/IIIa, a receptor for fibrinogen, and membrane skeletons in both unstimulated and thrombin-activated human platelets. After a treatment with dithiobis succinimidyl propionate (DTSP), a cross-linker, unstimulated and activated platelets were simultaneously extracted and fixed with a fixing solution containing Triton X-100. Also, the localization of GPIIb/IIIa on the plasma membrane was observed by a preembedding staining method of unextracted platelets. In unstimulated platelets, 20-40% of the whole plasma membrane remained in the detergent-extracted samples. Amorphous structures with 10-70 nm in diameters are distributed at 20 to 100-nm intervals on the surface of plasma membrane. Similar structures also were identified in the intact platelets by the immunocytochemical method. By careful inspection, we found that most of the amorphous structures that contained gold particles were connected to the submembrane zone just beneath the plasma membrane. The submembrane zone was identified as the membrane skeleton because actin was detected in the zone. After activation, detergent-insoluble granules were surrounded by dense networks of microfilaments in the central part of platelets. The filaments were identified as actin and became associated with myosin. These results demonstrate that GPIIb/IIIa on the plasma membrane is connected to the membrane skeleton and suggest that, during activation, actin filaments which extend into the cytoplasm from the membrane skeleton increase and form dense networks around Triton-insoluble granules.

Ultrastructural localization of lectin-binding sites on the surface of the guinea pig conjunctival epithelium

The cell-surface binding sites of two lectins, concanavalin A (Con A) and wheat-germ agglutinin (WGA) in the guinea pig conjunctiva were investigated at the ultrastructural level by means of pre-embedding staining of the tissue with lectin-colloidal gold complexes. Wheat-germ agglutinin, which recognizes N-acetylglucosamine and sialic acid residues, showed prominent and fairly uniform binding to the microvilli. The binding was markedly increased in the vicinity of the goblet cells, indicating that the same carbohydrate ligands were also present in the mucus. In contrast, no binding of concanavalin A, which recognizes mannose and N-glucose, was observed. The results suggest the presence of sialic acid and galactose as the constituent carbohydrates of glycoconjugates in the surface membrane of conjunctival epithelial cells as well as in the mucus produced by the goblet cells.

Ultrastructural immunocytochemical localization of excitatory amino acids in the somatosensory system.

We studied the ultrastructure and the synaptic arrangement of glutamate-immunoreactive terminals in rats, in the superficial laminae of the spinal cord, the brainstem cuneate nucleus, and the thalamic ventroposterolateral nucleus, where a role for glutamate as neurotransmitter has been suggested by biochemical, physiological and pharmacological approaches. The antiserum employed was raised against glutaramate conjugated to keyhole limpet hemocyanin with glutaraldehyde, and was used for pre-embedding staining with an avidin-biotin-peroxidase method and for post-embedding staining with an immunogold procedure. Both methods yielded similar results, consisting of labeling of selected terminals in all the areas examined. Double immunogold labeling on the same thin section using antisera against gamma-amino-butyric acid (GABA) or substance P (SP), in combination with the anti-glutamate serum, showed that staining for glutamate and GABA was present in different terminals in all the regions examined; glutamate and SP were co-localized in a few terminals only in the superficial laminae of the spinal cord. By performing immunogold staining in combination with anterograde tracing, glutamate immunoreactivity could be localized in identified primary afferents to the dorsal spinal cord and cuneate nucleus, and in lemniscal afferents to the thalamus.

A pre-embedding double staining procedure used to observe the effect of fixation on the activity of intercellular adhesion molecule on T and B cells

Intercellular adhesion molecule (ICAM-1) appears to be a ligand for LFA-1 dependent adhesion in T cell mediated cytotoxcity. It is found on cells of both hematopoietic and non-hematopoietic origin. While observing the activity of ICAM-1 on the surfaces of interacting T and B cells, we found that we could successfully carry out a pre-embedding double staining procedure utilizing both colloidal gold and peroxidase conjugated reagents.On 24-well microtiter plates, mitomycin-treated T4 cells were stimulated with 64.1 (anti-CD3) for one hour before the addition, in some instances, of B cells. Following a 12-48 hour incubation at 38°C, the cells were washed and then immunostained with a colloidal gold conjugated RFB-4 (anti-CD22); biotinylated R6.5 (anti-ICAM-1); followed by streptavidin/peroxidase. This method allowed us to observe two different antigens without concern about possible cross-reaction of reagents. Because we suspected ICAM-1 and R6.5 were sensitive to fixation, we tried varying concentrations of fresh paraformaldehyde before R6.5, after R6.5 and after streptavidin/peroxidase. All immunostaining and washing was done on ice with ice cold reagents.

Endocytosis and intracellular transport of colloidal gold-labeled ConA in mouse peritoneal macrophage

The purpose of this study is to investigate the endocytosis and intracellular transport of colloidal gold-labeled ConA (CG-ConA) in mouse peritoneal macrophages using pre-embedding staining method.Labeling with CG-ConA, acid phosphotase (AcPase) cytochemistry and electron microscope (EM). Mouse peritoneal macrophages were elicited by intraperitoneal injection of glycogen, after which 1ml CG-ConA (CG 1mg/ml) was injected per mouse. At different intervals after injection of CG-ConA (5,10,20 and 30 min), the peritoneal macrophages were isolated and treated for their AcPase activity according to the produce of Gomori. The CG-ConA and AcPase activity in these macrophages were examined under TEM.Surface distribution of CG-ConA 5-1Omin after intraperitoneal injection of CG-ConA many clusters and clumps of CG-ConA particles were formed on the macrophages (Fig. 1). The cluster formation is temperature-dependent and receptor-mediated, because the same phenomenon could be observed in the elicited macrophages after incubation with CG-ConA at 37°C in vitro, but not in those at 0-4°C and those incubated with pure CG particles. At 0-4°C CG-ConA particles were randomly and diffusely distributed over the cell surface. Aggregates of CG-ConA particles were often found between cellular processes.

Intracellular distribution of actin in cells of the orgen of Corti: A structural basis for cell shape and motility

Immunofluorescence staining and phalloidin labeling have provided localization of actin in the sensory and supporting cells of the inner ear at the light microscopic level. However, with electron microscopy, neither actin nor actin filaments have been found in the outer hair cell body. This paper describes various techniques utilized to preserve and identify cytoplasmic actin at the ultrastructural level. Post-embedding staining of Lowicryl K4M sections, pre-embedding staining of permeabilized cells of the organ of Corti, pre-embedding staining of vibratome sections, and pre-embedding staining of permeabilized dissociated cells documented the presence of actin, but each of these techniques was best suited to localize actin in specific parts of the cell. Cytoplasmic actin was labeled when isolated cells were lightly fixed and membranes were permeabilized with detergent--conditions under which the cell ultrastructure was compromised. Under conditions of optimal fixation, cytoplasmic filaments embedded in the dense granular matrix of the hair cell cytoplasm were observed.

Role of a 90-kD Glycoprotein in Heymann's Nephritis

Heymann's nephritis can be induced in rats by injection of antibodies (Ab) against the major nephritogenic antigen (Ag) (gp330). We previously reported on a 90-kD tubular-glomerular glycoprotein (gp90) having enzymatic activity (dipeptidyl-peptidase IV) which, after antibody interaction, induces a transient Heymann-like immunofluorescence pattern. In the present study we have analysed the ultrastructural features of the renal immune reaction occurring in rats after injection of a monoclonal antibody to gp90, revealed by a pre-embedding immunogold staining (5-nm particles). In the early stage (10-60 min) gold granules were found along the glomerular capillary walls mainly in the endothelial region. During the intermediate stage (24-48 h) the brush border microvilli were diffusely labelled. In the late stage (1-2 weeks) gold particles were still noticed at the tubular level. This work suggests that the immune reaction in Heymann's nephritis also occurs on the endothelial side of the glomerular capillary walls, thus facilitating the passage of epithelial-specific antibody. The tubular kinetics confirms that gp90 may play a role in the metabolic activity of tubular proximal cells. Because this antigen has been demonstrated in human kidney as well, it may be relevant to some membranous or other human nephropathies.

Immunoelectron microscopic localization of cell adhesion molecule L1 in developing rat pyramidal tract

The glycoprotein L1 is a cell adhesion molecule that has been proposed to function in the peripheral nervous system in axon fasciculation and onset of myelination. In this report we localize L1 during the development of a major central pathway: the pyramidal tract. The (sub)cellular localization of L1 was determined both by pre-embedding staining on Vibratome sections and by immunogold labelling on ultracryosections in developing rat pyramidal tract at the fifth cervical segment. On arrival at the fifth cervical segment, i.e. at postnatal day 1, growth cones of pioneer fibres did not exhibit L1-immunoreactivity. In the contact zone between pyramidal tract growth cones and glial processes no L1-immunoreactivity was observed. A clear L1-immunoreactivity was noted on small unmyelinated other axons situated in the entrance area of the pyramidal tract growth cones. Also on later arriving, i.e. between postnatal days 2 and 10, small unmyelinated fasciculating pyramidal tract axons L1 were present. It is our impression that L1 is localized in an irregular patchy way on the outer side of the axonal membrane. During the onset of myelination, i.e. between postnatal days 10 and 14, L1 could not be detected on axons ensheathed by oligodendrocytic processes. When myelination is largely completed, i.e. at postnatal day 21, the L1 antigen could be localized within the axoplasma of both unmyelinated and myelinated pyramidal tract axons. Furthermore, L1 could be observed occasionally on small unmyelinated pyramidal tract axons. Whereas compact myelin was always L1-negative, L1 was noted periaxonally between the axolemma and compact myelin and at (para)nodal regions at the contact zone between axolemma and oligodendrocytic processes. From these results it may be deduced that: (1) L1 is involved in fasciculation of outgrowing later arriving pyramidal tract fibres; (2) L1 is not involved in the onset of myelination in this central tract; (3) L1 might play an additional adhesive role in myelinated rat pyramidal tract.

Application of glial fibrillary acidic protein immunohistochemistry in the quantification of astrocytes in the rat brain.

Immunohistochemical staining for glial fibrillary acidic protein (GFAP) was employed as a tool for quantification of astrocytes in the rat brain. One-micron-methacrylate sections were prepared from 70-micron slices stained for GFAP by using a preembedding staining procedure. Numbers/unit area of astrocytes and nonastrocytes were determined for cortex, corpus callosum, and hippocampal neuropil. In each, counts from GFAP-stained, toluidine-blue-counterstained sections were compared with counts obtained from sections stained with toluidine blue alone. Numbers of nonastrocytes and total glia in all three regions were comparable in both groups of sections. Astrocyte counts in the cortex and hippocampus also showed no significant differences between the two groups. In contrast, the number of astrocytes in the corpus callosum was significantly lower in GFAP-stained, toluidine-blue-counterstained sections than in sections stained with toluidine blue alone. GFAP immunohistochemistry is a useful tool for the quantification of astrocytes in semi-thin plastic sections of rat brain.

Binding of a major secretory protein from bull seminal vesicles to bovine spermatozoa.

The seminal vesicles synthesize in an androgen-dependent manner a neutral protein of 13.5 kDa molecular weight that makes up about 40% of their secretion ("major protein"). An antiserum against this protein raised in rabbits was used to localize the antigen within the seminal vesicles. In addition to intraluminal secretion of the seminal vesicles and the ampulla of the vas deferens, ejaculated and ampullary spermatozoa revealed an intense immunoreaction, which was restricted to the neck region of the sperm head and the middle piece, while the principal piece of the tail as well as the sperm head were devoid of immunoreactive material. Comparison of spermatozoa taken from the tail of the epididymis with ampullary spermatozoa showed that about 90% of the latter, but only 10-20% of the former presented this distributional pattern of immunoreactive sites. Epididymal epithelium as well as calf seminal vesicle epithelium showed no immunoreactivity with major protein antiserum. Using a pre-embedding staining technique with gold-labeled primary or secondary antibodies, respectively, no immunostaining could be achieved at the ultrastructural level. Incubation experiments of epididymal spermatozoa in EGTA-containing solutions in the absence of calcium resulted in a gradual labilization and eventual loss of the plasma membrane of the sperm middle piece. After removal of (at least part of) the plasma membrane, bound major protein could be visualized immunohistochemically close to the mitochondria of the middle piece using a gold-labeled primary or secondary antibody. The acceptor site for major protein therefore seems to reside inside the plasma membrane of the sperm middle piece.(ABSTRACT TRUNCATED AT 250 WORDS)

Gabaergic innervation of somatostatin-containing neurosecretory cells of the anterior periventricular hypothalamic area: A light and electron microscopy double imunolabelling study

Double immunolabelling on semithin sections revealed glutamate decarboxylase immunopositive dots surrounding somatostatin-containing cell sections in the rat periventricular hypothalamic area. Up to 12 appositions were observed per cell section with an average number of 2 3 and a unimodal distribution. At the electron microscopical level pre-embedding staining of glutamate decarboxylase showed that most immunoreactive elements consisted of immunolabelled axonal endings. Most of these glutamate decarboxylase immunopositive boutons were found within the neuropil where they frequently made synapses on unidentified dendrites. Some of them were apposed to somatostatin-containing cell bodies that were identified according to the presence of immunolabelled granules using combined immunogold post-embedding staining. In many instances glutamate decarboxylase immunoreactive endings were also found to be involved in synaptic contact with somatostatin-labelled perikarya. or neuronal processes. These contacts provide the morphological basis for a direct GABAergic control of the somatostatincontaining cells regulating the secretion of growth hormone.

Ultrastructural detection of carbohydrates in the pellicle of Pneumocystis carinii

The presence of carbohydrates in the pellicle of Pneumocystis carinii was demonstrated by methenamine silver (MS) and thiocarbohydrazide-silver proteinate (TCH-SP) staining techniques. An intense, positive reaction with MS was observed on both the electron-dense outer layer and the electron-lucent middle layer of the pellicle, whereas in the case of TCH-SP only the outer layer stained well. The middle layer did not stain as intensely with TCH-SP as with MS. This observation indicates that the pellicle of P. carinii contains carbohydrates but that the outer and middle layers differ in carbohydrate composition. The binding sites of concanavalin A (Con A) and Macura pomifera (MPA) lectins were elucidated on the pellicle of P. carinii using colloidal gold as a marker. In a preembedding staining method using Con A and MPA lectins, the adherence of gold particles was observed on the surface of all stages of the parasite. The homogeneous distribution of the gold particles indicates that the outer electron-dense layer contains both Con A- and MPA-specific carbohydrates. In postembedding staining with Con A, the adherence of gold particles was found on both the outer and middle layers, whereas in the case of MPA only the outer layer was labeled with gold particles. These results indicate that the electron-dense outer layer contains a considerable amount of carbohydrates specific for these lectins, whereas the electron-lucent middle layer seems to lack MPA-specific carbohydrates.

Specialization of the interphotoreceptor matrices around cone and rod photoreceptor cells in the monkey retina, as revealed by lectin cytochemistry

The binding sites of two lectins, peanut agglutinin (PNA) and wheat germ agglutinin (WGA), in the interphotoreceptor matrix (IPM) and photoreceptor plasma membranes of the Japanese monkey (Macaca fuscata) retina were localized using a pre-embedding staining method with ferritin-conjugated (Fer) lectins as well as a postembedding staining method with fluorescence-labeled (FITC) lectins. FITC-PNA, but not WGA, stained cylindrical domains of the IPM around cone outer and inner segments, while the IPM around rods stained with FITC-WGA but not PNA. When the intact (not detached) retinal tissues were incubated with Fer-lectin, the lectin generally labeled neither the IPM nor photoreceptor plasma membranes, but labeled only those structures in detached portions occurring at the edges of occasional retinal tissue blocks. Thus, the neural retinas physically isolated from the retinal pigment epithelium (RPE) were utilized principally here. Ultrastructurally, the IPM in the intact retina consisted of granular and filamentous materials; the IPM in the isolated neutral retina also retained those components, although somewhat loosely organized, and the IPM around cones appeared to be preserved better than did the IPM around rods. Fer-PNA bound to the IPM associated with cones, but not rods; Fer-WGA bound to the rod- but not cone-associated IPM. The ferritin particles were found to lie close to the granular and filamentous materials. Those photoreceptor-associated IPMs extended to the apical surface of the RPE in detached portions or to the apical villi of the RPE which were frequently found in the isolated neural retinas. Also, Fer-PNA labeled the cone, but not rod, plasma membranes; Fer-WGA bound heavily to the plasma membranes of rod and cone outer segments, but sparsely to those of their inner segments. These results suggest that the IPM comprises chemically and physically differential domains specialized for cone and rod photoreceptor cells, and that these specialized IPM are structurally so stable that may be involved in isolating photoreceptor cells physicochemically from each other and in the interactions between the photoreceptors and the RPE, such as retinal adhesion.

Immunocytochemical staining of isolated rat Sertoli cells for anti-FSH beta.

Sertoli cells are a primary target for the action of follicle-stimulating hormone (FSH) in the testis. The purpose of this investigation was to verify ultrastructurally that FSH binds to receptors on the plasma membrane of isolated rat Sertoli cells. A relatively pure aliquot of Sertoli cells was obtained by first dissociating testicular tissue from immature rats with collagenase and then centrifuging the suspension in Percoll density gradients. Pre-embedding staining with the peroxidase anti-peroxidase (PAP) complex technique using anti-FSH beta as the primary antiserum localized endogenous receptor-bound FSH on the plasma membrane of isolated Sertoli cells. Staining was considered to be specific since membranes of Sertoli cells derived from hypophysectomized rats were not stained when subjected to the same procedure. Cytoplasmic vesicles in Sertoli cells from experimental, control, and hypophysectomized groups also stained with PAP. Staining of these structures appeared to be specific since it was obliterated by preabsorption of anti-FSH beta with FSH. Preabsorption with luteinizing hormone (LH) did not affect the staining of cytoplasmic vesicles. The results of this investigation provide the first evidence for ultrastructural localization of specific binding sites for anti-FSH beta on the cell membrane of isolated Sertoli cells using an unlabeled antibody technique, and they further support the contention that Sertoli cells are a primary target for the action of FSH.

Application of lectin--gold complexes for electron microscopic localization of glycoconjugates on thin sections.

A method is described for the electron microscopic detection of lectin-binding sites in different cellular compartments and extracellular structures that uses thin sections from resin-embedded tissues. Various lectins (Ricinus communis lectin I and II, peanut lectin, Lotus tetragonolobus lectin, Ulex europeus lectin I, Lens culinaris lectin, Helix pomatia lectin, and soybean lectin) were bound to particles of colloidal gold and used for direct staining of thin sections or glycoprotein--gold complexes were prepared and applied in an indirect technique (concanavalin A and horseradish peroxidase--gold complex; wheat germ lectin and ovomucoid--gold complex). The details for preparation of such complexes from 14 nm gold particles are reported. The conditions of tissue processing that gave satisfactory staining results and good fine structure preservation were mild aldehyde fixation without osmification and low temperature embedding with the hydrophilic resin Lowicryl K4M. None of the so-called etching procedures was necessary prior to labeling of Lowicryl K4M thin sections. Examples of the use of this approach for detection of glycoconjugates in the rough endoplasmic reticulum, Golgi apparatus, and mucin of intestinal goblet cells as well as plasma membrane and various intracellular structures of absorptive intestinal and renal tubular cells are shown. A comparison is made with preembedding staining results on Concanavalin A-binding site localization in rat liver which shows that problems of penetration common in such a technique are circumvented by the postembedding approach described here. Concanavalin A-binding sites were not only consistently found in nuclear envelope, rough and smooth endoplasmic reticulum, plasma membranes, and collagen fibers, but also in mitochondria, glycogen, ribosomes, and nucleus. These data and those of a previous investigation (Roth J, Cytochem 31:547, 1983) prove the applicability of this cytochemical technique for postembedding localization of glycoconjugates by light and electron microscopy.

Light- and electron-microscopic immunocytochemistry of glutamic acid decarboxylase (GAD) in the basal hypothalamus: Morphological evidence for neuroendocrine γ -aminobutyrate (GABA)

GABAergic cells and axon terminals were localized in the basal hypothalamus of different species (rat, mouse and cat), by means of an immunocytochemical approach using a specific and well-characterized antiserum to the GABA biosynthetic enzyme, glutamate decarboxylase. Lightmicroscopic visualization was performed with an indirect immunofluorescence method and electron-microscopic observations were made on material with pre-embedding staining and use of the peroxidase-antiperoxidase procedure. At the light-microscopic level, a dense immunofluorescent plexus was observed over both the medial and lateral parts of the external layer of the median eminence. The labelling extended from the rostral part of the median eminence up to the pituitary stalk. Over the subependymal and internal layers only a few immunoreactive dots were visible, except around the blood vessels where they appeared more concentrated. Immunoreactive varicosities could be found following the outlines of the capillary loops and lining tanycyte processes, especially in the median eminance midportion. At the electron-microscopic level, the immunolabelling was exclusively found over neuronal profiles in the median eminence. The latter represented a small fraction of the total number of varicosities visible on the same section. Labelled profiles typically contained numerous small clear synaptic vesicles and only a few or no dense-core vesicles. In the subependymal and internal layers, rare labelled endings were found close to ependymal cells or among transversally cut fibers, respectively. In the palisadic zone, elongated positive boutons were visible intermingled with bundles of unlabelled axons and glial or ependymal processes. In the neurohemal contact zone, immunoreactive endings were observed among unlabelled neurosecretory endings in close vicinity to fenestrated capillary perivascular space. Small moderately intense immunofluorescent varicosities were observed all over the hypothalamus. The density of the glutamate decarboxylase-positive network was higher than in most diencephalic regions. Intraventricular or topical injection of colchicine allowed the visualization of small lightly immunoreactive cells in the diffusion area of colchicine. In the arcuate nucleus labelled axonal endings containing small pleomorphic synaptic vesicles and sometimes a few dense-core vesicles were observed at the electron-microscopic level. Typical synaptic junctions were commonly found between positive endings and unlabelled perikarya, or more frequently, unlabelled dendrites. These findings show that glutamate decarboxylase-containing endings are localized in several strategic sites for potential GABAergic neuroendocrine regulations. The GABAergic endings found among neurosecretory endings in the neurohemal contact zone may provide the morphological support for the release of γ -aminobutyrate into the portal blood flow as an hypothalamic hypophysiotropic hormone. Alternatively, neurosecretory cells might be under GABAergic control expressed either at their terminal level within the median eminence or the cell body level within the parvicellular hypothalamic nuclei.

Pre-embedding immunohistochemistry as an approach to high resolution antigen localization at the light microscopic level.

Pre-embedding immunohistochemistry with subsequent embedding in hydroxypropyl methacrylate enables one to obtain high resolution staining of antigens in 1 mu tissue sections. A routine method using formaldehyde fixation, methanol permeation, and an indirect method with fluorescein-labeled second antibody is described. This method is compared with other pre-embedding staining procedures. To illustrate the method the mouse small intestine was chosen as a model and stained with antibodies to tubulin, actin, and fibronectin. Some anticipated and some unusual staining patterns were found.

Immunocytochemical demonstration of vasopressin and oxytocin in the rat brain by light and electron microscopy.

Vasopressin and oxytocin were demonstrated by means of the unlabeled antibody enzyme method in the rat central nervous system. Vasopressin and oxytocin fibers of the magnocellular nuclei were found to run towards the amygdala, hippocampus, and spinal cord. From the parvocellular suprachiasmatic nucleus only vasopressin containing fibers were found to run towards the organum vasculosum lamina terminalis, lateral septum, and lateral habenular nucleus. Immunoelectron microscopy, using the preembedding staining technique, frequently demonstrated vasopressin-containing synapses on neuronal structures in the lateral septum, lateral habenular nucleus, and amygdala. These results suggest a putative neurotransmitter role for these neuropeptides.

Immuno-electron microscopical demonstration of vasopressin and oxytocin synapses in the limbic system of the rat

The extensive distribution of exohypothalamic vasopressin or oxytocin containing nerve fibres is thought to be the anatomical basis for the involvement of these neuropeptides in central processes. Following light microscopic observations suggesting that these fibres terminate on other neurons, the present study was undertaken to demonstrate the existence of such endings in the limbic system, which is one of the main target areas for these peptides. For immunoelectron microscopy glutaraldehyde-paraformaldehyde perfused brains of male Wistar rats and Brattleboro rats, homozygous for diabetes insipidus, with and without postfixation in OsO4, were used. Post-embedding staining revealed false positive reaction product on all dense core vesicles, e.g., in the lateral septum. With pre-embedding staining, however, intense and specific reactions were observed for both vasopressin and oxytocin at their sites of production, as well as the neurohypophysis and in the extrahypothalamic limbic brain regions. In the lateral septum and habenular nucleus only vasopressin-containing synapses could be demonstrated, while in the medial nucleus of the amygdala synapses containing either vasopressin or oxytocin were observed. These peptide containing synapses do not seem to differ in any fundamental way from the classical transmitter-containing synapses in the brain.

Localization of neurophysin within organelles associated with protein synthesis and packaging in the hypothalamoneurohypophysial system: an immunocytochemical study.

Electron microscopic immunocytochemical localization of neurophysin in the hypothalamo-neurohypophysial system of mice has been studied by using the pre-embedding staining approach to the unlabeled antibody-enzyme technique of Sternberger. In supraoptic cell bodies, peroxidase-antiperoxidase reaction product was localized within cisternae of the nuclear envelope, rough endoplasmic reticulum, and Golgi saccules but not in GERL (Golgi-associated smooth endoplasmic reticulum from which lysosomes arise). Reaction product was also present in secondary lysosomes. Secretory granules in supraoptic perikarya and posterior pituitary Herring bodies were likewise immunoreactive. These findings with the unlabeled antibody-enzyme technique provide conclusive evidence for the localization of an antigen within cellular organelles associated with protein synthesis and packaging.