Electron Microscopic Immunocytochemical Study Research Articles

Histaminergic axons in the neostriatum and cerebral cortex of the rat: a correlated light and electron microscopic immunocytochemical study using histidine decar☐ylase as a marker

Histaminergic nerve fibers and their axonal varicosities in the neostriatum and cerebral cortex were light and electron microscopically examined by means of peroxidase-antiperoxidase immunocytochemistry with histidine decar☐ylase (HDC) as a marker. A majority of HDC-like immunoreactive axonal varicosities observed in serial thin sections for electron microscopy exhibited no synaptic contacts in either the neostriatum or cerebral cortex. The remaining small proportion of immunoreactive axonal varicosities formed synaptic contacts with non-immunoreactice dendritic shafts and spines.

Purification of membrane polypeptides of rat liver peroxisomes.

Peroxisomes were obtained by sucrose density gradient centrifugation from the livers of di(2-ethylhexyl)phthalate-fed rats, and the membranes were prepared by carbonate extraction (Fujiki, Y., Fowler, S., Shio, H., Hubbard, A.L., & Lazarow, P.B. (1982) J. Cell Biol. 93, 103-110). The integrated membrane polypeptides were solubilized with sodium dodecyl sulfate, and purified by repeated polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Separation of 70 and 68 kDa polypeptides was not attempted in the present study because of their close migration in polyacrylamide gel electrophoresis. Other polypeptides with apparent molecular masses of 41, 27, 26, and 22 kDa were purified to near homogeneity. Antibodies were raised against these purified preparations. The 68 kDa polypeptide is suggested to be produced by the proteolytic modification of 70 kDa polypeptide, since the former increased concomitantly with decrease of the latter when the liver homogenate was incubated, and this change was prevented in the presence of leupeptin during the incubation. The 41 kDa polypeptide was a minor component. The 70 and 68 kDa polypeptides and 41 kDa polypeptide and their antibodies were cross-reactive, but the relation of these polypeptides was not clear. The 27 and 26 kDa polypeptides seemed to be another species of membrane polypeptides, although the relationship of these two polypeptides remains to be clarified. The 22 kDa polypeptide is not related to other membrane polypeptides. The results of immunoblot analysis of subcellular fractions of the liver and an electron microscopic immunocytochemical study to locate the antigenic sites with protein A-gold complex suggest that all of these polypeptides are localized on peroxisomal membranes. On proliferation of rat liver peroxisomes by administration of di(2-ethylhexyl)phthalate, a peroxisome proliferator, all of these polypeptides were markedly increased.

Electron microscopic immunocytochemical study of somatostatin neurons in the periventricular nucleus of the rat hypothalamus with special reference to their relationships with homologous neuronal processes

The neurons containing somatostatin in the rat periventricular nucleus were studied by using a modified electron microscopic immunocytochemical technique that improves both the penetration of immunoreagents into unembedded immunostained tissues and the preservation of ultrastructural morphology. Inside perikarya and dendrites, immunostaining was not only associated with neurosecretory granules but also with ribosomes and saccules of the cis face of the Golgi apparatus. In the axonal profiles found in this region the labeling was observed both on neurosecretory granule cores and on the limiting membrane of small synaptic-like vesicles. Throughout the periventricular nucleus, both non-synaptic and synaptic relationships were shown between labeled neurons. Non-synaptic relationships mainly consisted of direct apposition of the membranes of neighboring neurons by dendrosomatic, somasomatic or dendrodendritic contacts. These labeled perikarya and dendrites were also synaptically contacted by labeled axonal endings containing numerous aggregated synaptic-like vesicles. The physiological significance of the synaptic and non-synaptic relationships between somatostatinergic neurons is discussed in terms of possible synchronization between homologous neurons of the somatostatin neuroendocrine system and control of these neurons by a central ultra-short loop feedback mechanism.

Light and electron microscopic immunocytochemistry of the same nerves from whole mount preparations.

A technique for performing correlated light and electron microscopic immunocytochemical studies on whole mount preparations has been developed using myenteric plexus from guinea pig small intestine as a model. With this method a structure containing a particular antigen can first be located by light microscopy and then examined with the electron microscope. Pieces of intestine pinned on balsa were incubated in oxygenated Krebs solution at 37 degrees C for 90-120 min and then fixed for 1 hr at room temperature in 4% formaldehyde, 0.05% glutaraldehyde, and 0.2% picric acid in 0.1 M sodium phosphate buffer, pH 7.4. The tissue was washed vigorously in several changes of 50% ethanol until the picric acid had been removed, stored overnight in phosphate buffer, and then exposed to 0.1% sodium cyanoborohydride in buffer for 30 min. Vasoactive intestinal peptide (VIP) was localized in separated layers containing myenteric plexus and longitudinal muscle using the peroxidase-antiperoxidase technique with imidazole intensification of the diaminobenzidine reaction product. At the light microscope level, tissue stained by this technique showed VIP-immunoreactive nerve cell bodies and processes throughout the thickness of the myenteric ganglia in numbers approximately equivalent to those seen in whole mounts processed by an established technique for the light microscopic demonstration of VIP, which does not involve exposure of tissue to glutaraldehyde. VIP-immunoreactive structures that were first identified at the light microscope level were subsequently examined at the electron microscope level. VIP-immunoreactive axon profiles were found to form synapses on both immunoreactive and nonimmunoreactive myenteric neurons. The fine structural appearance of the different cell types present in whole mount preparations prepared by this method was similar to that seen in conventionally fixed tissue, except that free and bound ribosomes were absent from the tissue processed for immunocytochemistry. The method described here is reliable and no more difficult than presently available methods for preembedding electron microscopic immunocytochemistry on sections. Its main advantage is that immunoreactive structures for ultrastructural study can be selected from the entire population of chemically identified nerves within a whole mount rather than from a smaller sample present within a section. This technique is applicable to other tissues that can be stained immunohistochemically in whole mounts. The fixation and penetration enhancement procedures can also be adapted for immunocytochemical studies on vibratome or frozen sections.

Co-localization of corticotropin-releasing factor and vasopressin in median eminence neurosecretory vesicles.

Vasopressin (VP) potentiates the effect of corticotropin-releasing factor (CRF) on the secretion of adrenocorticotropic hormone (ACTH) from anterior pituitary cells in vitro, and both CRF and VP have been found in portal blood. These data support the hypothesis that VP acts synergistically with CRF to cause the secretion of ACTH in vivo but the origin of the CRF and VP, and the physiology of their release, have not been precisely defined. Parvocellular cell bodies in the paraventricular nucleus (PVN) which project to the external zone of the median eminence can be stained for both CRF and VP after adrenalectomy, and there is light microscopic immunocytochemical evidence that neurophysin (NP) may be located within some of the CRF-containing axons. Electron microscopic immunocytochemical studies have demonstrated the presence of CRF, VP and its 'carrier' protein, VP-associated neurophysin (NP-VP) in 100-nm neurosecretory vesicles (NSVs) in axons terminating near the portal capillary plexus in the external zone of the median eminence. If these peptides are extensively co-localized in the same NSVs in the median eminence, then coordinate secretion of CRF and VP in vivo is obligatory, at least in some physiological circumstances. We demonstrate in this report, using post-embedding electron microscopic immunocytochemistry on serial ultrathin sections, that CRF, VP and NP-VP are contained not only in the same axons and terminals, but in the same 100-nm NSVs in the median eminence of both normal and adrenalectomized rats. In addition, in the normal rat median eminence 44% of the CRF-positive axons and terminals stained strongly for VP and NP-VP, whereas in the adrenalectomized rat virtually all the CRF-positive structures in the median eminence showed strong staining for VP and NP-VP, indicating a transformation of one subpopulation of CRF-positive axons and terminals by adrenalectomy.

Electron Microscopic Immunocytochemical Localization of Myelin Proteolipid Protein and Myelin Basic Protein to Oligodendrocytes in Rat Brain During Myelination

Electron microscopic immunocytochemical studies were carried out to localize myelin basic protein and myelin proteolipid protein during the active period of myelination in the developing rat brain using antisera to purified rat brain myelin proteolipid protein and large basic protein. The anti-large basic protein serum was shown by the immunoblot technique to cross-react with all five forms of basic protein present in the myelin of 8-day-old rat brain. Basic protein was localized diffusely in oligodendrocytes and their processes at very early stages in myelination. The immunostaining for basic protein was not specifically associated with any subcellular structures or organelles. The ultrastructural localization of basic protein suggests that it may be involved in fusion of the cytoplasmic faces of the oligodendrocyte processes during compaction of myelin. Immunoreactivity in the oligodendrocyte and myelin due to proteolipid protein appeared at a later stage of myelination than did that due to basic protein. Staining for proteolipid protein in the oligodendrocyte was restricted to the membranes of the rough endoplasmic reticulum, the Golgi apparatus, and apparent Golgi vesicles. The early, uncompacted periaxonal wrappings of oligodendrocyte processes were well stained with antiserum to large basic protein whereas staining for proteolipid protein was visible only after the compaction of myelin sheaths had begun. Our evidence indicates that basic protein and proteolipid protein are processed differently by the oligodendrocytes with regard to their subcellular localization and their time of appearance in the developing myelin sheath.

Estrogen responsive cells in the arcuate nucleus of the rat contain glutamic acid decar☐ylase (GAD): an electron microscopic immunocytochemical study

Twenty-one days after ovariectomy (OVX) an increased frequency of lamellar cytoplasmic organelles, termed ‘whorl’ bodies (WB), was observed in neurons of the rat arcuate nucleus (AN). When estradiol valerate (2 mg s.c.) was injected either at the time of OVX or one week later, the frequency of WB at 21 days was reduced. The estrogen treatment resulted in a concomitant rise in the frequency of ‘nematosomes’ (NS), filamentous electron-dense cytoplasmic structures. In the medial part of the AN glutamic acid decar☐ylase (GAD) immunopositive symmetric (Gray II) synapse were observed in contact with WB- and NS-containing cells. After colchicine treatment, GAD immunoreactivity was observed in the WB- and NS-containing perikarya in the medial AN. Some of the NS-containing cells in the lateral AN of the colchicine-pretreated animals remained immunonegative for GAD.

Immunocytochemical localization of somatostatin-28 1–12 in the rat hypothalamus

Somatostatin-28 (SS28) is considered as a precursor of somatostatin-14 (SS14) and also of the remaining NH 2 terminus of SS28, the dodecapeptide SS28 1−12 which has been recently characterized. In order to study the cellular and subcellular localizations of SS28 1−12 in the rat hypothalamus, we have conducted a light and electron microscopic immunocytochemical study, using both the peroxidase-antiperoxidase and the immunogold technique. Several antisera which selectively recognize one or more of these 3 somtostatin-related peptides were used. In serial paraffin sections, it was observed that SS28 1−12 was contained in the same neuronal cell bodies which also contain SS28. These neurons were exclusively located in the previntricualr nucleus. All the antisera used also produced a strong staining in the external zone of the median eminence. At the electron microscopic level, the 3 peptides were exclusively localized in all the dense core vesicles in cell bodies and axons in the periventricular nucleus and terminals in the median eminence. These results strongly suggest that SS28 1−12 and SS14 originate from the precursor SS28 and that processing of the precursor occurs in the cell body. They also support the hypothesis that the 3 peptides are released simultaneously under appropriate stimulation.

Preoptic LH-RH and somatostatin in the rat median eminence. An experimental light and electron microscopic immunocytochemical study.

Glass microknife lesions and immunocytochemistry were used to evaluate luteinizing hormone-releasing hormone (LH-RH)- and somatostatin (SS)-immunoreactive pathways from the preoptic region to the rat median eminence. Cuts were so placed that axons of more caudally located neurons in the periventricular hypothalamic areas were spared. Light and EM observations of LH-RH-immunostained preparations indicated that following the midline periventricular cuts the density of LH-RH labelled axons and axon terminals in the ME appeared similar to that of nonlesioned animals. Following bilateral lateral hypothalamic cuts placed between the preoptic area and the ME, LH-RH immunostaining in the ME was markedly reduced. This provides evidence that the preponderance of LH-RH axons originating from the preoptic area reach the ME by a lateral hypothalamic route. In contrast to the LH-RH findings, midline lesions made using the same coordinates caused a noticeable reduction in SS immunostaining in the accurate nucleus and ME. There was either no change or only minimal change after the lateral cut. Somatostatin axons arising from the preoptic periventricular nucleus take a periventricular route and contribute to median eminence innervation, but much less extensively than the more caudally located somatostatin neurons in the hypothalamic periventricular nucleus [19].

Preparation and discharge of secretion in the subcommissural organ of the rat. An electron-microscopic immunocytochemical study.

The secretion of the subcommissural organ (SCO) of the rat was studied by means of immunocytochemistry at the electron-microscopic level with the use of (1) the polar embedding medium Lowicryl K4M at -30 degrees C, (2) the protein A-gold technique, and (3) a rabbit antiserum against bovine Reissner's fiber (see Sterba et al. 1981). Two different substructures of the ependymal and the hypendymal SCO-cells display a positive immunocytochemical reaction: (1) sacs containing flocculent secretion, which originate from the granular endoplasmic reticulum, and (2) vacuoles filled with fine granular secretion, which are pinched off from the Golgi apparatus. The secretory material of the sacs and the vacuoles is discharged both (i) apically into the cerebrospinal fluid and (ii) basally into intercellular spaces of the SCO-hypendyma. The apically released secretion is condensed to a lamina-like formation, which more caudally assumes the form of Reissner's fiber. The route of the basally released secretion remains, however, vague. The "periodically striated bodies", which were thought to be morphological mediators of the discharge of the secretion into the capillaries, are never labeled by gold particles.

Immunocytochemical localization of alpha-D-mannosidase II in the Golgi apparatus of rat liver.

Mannosidase II is involved in the trimming of alpha-1,6-mannosyl residues during the biosynthesis of glycoproteins containing N-linked oligosaccharides of the complex type. A highly specific polyclonal antibody (IgG) was isolated from rabbits immunized with a homogeneous preparation of mannosidase II prepared from rat liver. With this antibody, light and electron microscopic immunocytochemical studies on rat liver reveal that essentially all mannosidase II in hepatocytes is localized in the Golgi apparatus, the only other site with reaction product being the endoplasmic reticulum. The indirect immunocytochemical method used in this study involved three major steps: exposure of aldehyde-fixed tissue to immune and nonimmune IgG, treatment with staphylococcal protein A labeled with horseradish peroxidase, and incubation in diaminobenzidine to reveal sites of peroxidase activity. The procedures described overcome major problems in immunocytochemistry, allowing preservation of antigenic sites and maintaining adequate ultrastructural integrity. The in situ localization of other carbohydrate-processing enzymes, involved in either trimming or attachment of sugar residues, should be possible with this procedure. Because biosynthetic precursors of the processing enzymes may be revealed by an immunocytochemical approach, it is potentially significant that mannosidase II reaction product is present in areas of the endoplasmic reticulum as well as in the Golgi apparatus.

Experimental Herpesvirus Keratitis in the Rabbit: Topical versus Intrastromal Infection Routes

Intrastromal and topical routes of infection of rabbit corneas with the HF strain of herpes simplex virus type 1 were compared clinically to determine which route of infection would present the best model of disciform edema and of deep stromal keratitis for our further studies on how the immune response of the infected animals may influence the expression of clinical disease. In addition, virus clearing and persistence of viral antigens as immunologic stimuli were monitored by virologic and electron microscopic immunocytochemical studies. We found topical infection to be preferable to the intrastromal infection route for presenting stromal disease in that it resulted in a higher incidence of epithelial disease with less anterior chamber involvement. The topical infection route model should be valuable in studies of cell-mediated immune response against HSV-infected cells.

Surface labelling of oligodendrocytes with anti-myelin serum in cell cultures from the rat brain. Light- and electron-microscopic immunocytochemical studies.

Antiserum produced in rabbits against purified myelin contains antibodies that bind to the surface of cells having multiple branched processes in live cultures of cerebral hemispheres of newborn rats. The identity of these cells was determined by double immunolabelling experiments with other specific antigenic markers (W1 Wolfgram protein, myelin basic proteins, glial fibrillary acidic protein). It was demonstrated that the cells are a subclass of oligodendrocytes which have reached a certain degree of maturation; nearly all of them contain basic proteins. Indeed, a number of oligodendrocytes, that contain the W1 protein, and may or may not have processes, are not surface-labelled in similar conditions. The usefulness of such an anti-myelin serum in the isolation of pure oligodendrocytes is discussed.

The occurrence of albumin in the rat liver. A light and electron microscope immunocytochemical study

The occurrence of albumin in the rat liver. A light and electron microscope immunocytochemical study

Stria terminalis axons ending on substance P and neurotensin-containing cells of rat central amygdaloid nucleus: An electron microscopic immunocytochemical study

Immunocytochemistry at the electronmicroscopic level was used to identify and examine substance P and neurotensin-containing perikarya and dendrites in the central amygdaloid nucleus of the rat. Following unilateral transection of the stria terminalis between the bed nucleus of the stria terminalis and the amygdala, degenerated nerve terminals were present in the ipsilateral central amygdaloid nucleus. These degenerated boutons were associated with both perikarya and dendrites of substance P and neurotensin-positive cells as well as unlabeled neurons.

Physiology of nerve growth factor.

Physiology of nerve growth factor.

Electronmicroscopic immunocytochemical study on the vasopressin-containing neurons of the thirsting rat.

Whereas in thirsting animals the perikarya of the nucleus supraopticus are nearly empty of neurosecretory granules as evidenced by electron microscopic observation, the perikarya are heavily stained by light microscopic immunohistochemical staining. In an attempt to discover the substrate responsible for the positive immunohistochemical staining in thirsting rats, the neurons of the supraoptic nucleus of normal and long-term thirsting animals were compared by electron microscopic immunocytochemistry (indirect PAP-method). In controls all parts of the vasopressin-synthesizing neuron are filled with elementary granules which render a positive and uniform reaction after immunostaining with the indirect PAP-method. The positively reacting fibers in the external zone of the median eminence contain smaller granules than those of the tractus supraoptico-hypophyseus. Within the nucleus suprachiasmaticus, no positive reaction after immunostaining was found. In long-term thirsting animals PAP-complexes as markers of vasopressin are located over the ergastoplasm and over the few small elementary granules. The processes within the nucleus supraopticus and the ballooned axons in the internal zone of the median eminence exhibit “free”, i.e. non granule-bound, PAP-complexes. Findings in the nucleus suprachiasmaticus and the median eminence of thirsting animals correspond to those in controls. The neurohypophysis is almost completely devoid of PAP-labeled elementary granules.

Immunocytochemical evidence for particulate localization of phenylethanolamine- N-methyltransferase in adrenal medulla

Previous biochemical evidence for the localization of phenylethanolamine- N-methyltransferase in adrenal medulla has indicated that this enzyme is soluble, although some workers have reported a significant amount of particulate phenylethanolamine- N-methyltransferase (Joh and Goldstein, 1973). Electron microscopic immunocytochemical study of bovine adrenal medulla has revealed a distinct particulate localization of phenylethanolamine- N-methyltransferase in association with chromaffin granules. It is therefore suggested that some, and possibly most, adrenal phenylethanolamine- N-methyltransferase may be in a particulate form in intact tissue, and that solubilization artifacts during tissue preparation could account in part for the large proportion of soluble phenylethanolarmine- N-methyltransferase reported previously.

Corticotroph cells in primary cultures of rat adenohypophysis: A light and electron microscopic immunocytochemical study

Monolayer cultures of trypsin-dispersed cells of the rat adenohypophysis were grown for 5 to 54 days. ACTH was localized by immunocytochemistry using an antiserum to synthetic ACTH1-28 prepared in rabbit and sheep anti-goat immunoglobulin coupled with peroxidase. ACTH content of the culture medium was measured by radioimmunoassay. Corttion time. The corticotrophs retained their essential morphological characteristics. Immunological staining was found in the secretory granules, some tubular or saccular structures, parts of the rough endoplasmic reticulum, and the cytoplasmic matrix. Immature secretory granules in the Golgi apparatus as well as some Golgi elements showed different degrees of immunoreactivity. In agreement with the high ACTH content of the culture medium the number, size and shape of the secretory granules, the active Golgi apparatus, the high amount of extragranular ACTH as well as pictures suggesting granule extrusion claim for a high ACTH synthesis and transport (and low ACTH storage) in the cultured corticotrophs.