Basic and applied histochemistry最新文献

A histochemical investigation was carried out on proteoglycans of bovine intervertebral disc. Samples obtained from the "annulus fibrosus" (A.F.) and "nucleus pulposus" (N.P.) were treated with Alcian blue (AB) diluted in solutions of MgCl2 at critical electrolyte concentrations (CEC); some samples were incubated in testicular hyaluronidase before AB treatment. At least four types of elongated AB-proteoglycan particles were recognized: a) in A.F. lamellae and N.P., 1 nm rod-like particles were arranged orthogonally to the collagen fibrils and spaced at a distance equivalent to the fibril D-period (Figs. 1-5); b) within the A.F. lamellae, other 16-20 nm particles formed a close network among the collagen fibrils (Figs. 1,2,3,5); c) in the A.F. interlamellar crevices, 30-50 nm leaf-like particles were present (Fig. 6); d) in the N.P.Z., 20-30 nm leaf-like particles formed a wide-mesh (Fig. 4). The alcianophylic particle sizes suggest they may correspond to proteoglycan monomers in the A.F. lamellae and mostly proteoglycan aggregates in A.F. interlamellar crevices and N.P.. Both alcianophylia degrees at MgCl2 CEC solutions and enzymatic susceptibility indicate the presence of chondroitin sulphate and keratan sulphate and that the large particles in the A.F. interlamellar crevices are the keratan sulphate richest proteoglycans. The features of the observed AB-proteoglycan particles are consistent with previous morphological data reported for other tissues as well as some biochemical data for the intervertebral disc and may be correlated to the composite mechanical properties of this tissue.

Immunohistochemical analysis of the extrahypothalamic distribution of vasotocin-like immunoreactive elements within the central nervous system of the domestic fowl and Japanese quail, revealed several mesencephalic, pontine and bulbar target areas topographically identifiable. Extrahypothalamic immunopositive perikarya were observed in diencephalic and mesencephalic locations after glutaraldehyde fixation.

A study was made of the distribution of Substance P-immunoreactive fibers in chick cornea and uvea in whole mount preparation, using the indirect immunofluorescence technique. The development of these fibers during embryogenesis was also investigated. SP-fibers were present in all chick eye structures, in the various prenatal and postnatal stages examined. Their distribution was comparable with that observed by other workers in mammals. Transformation of the iris musculature from smooth to striated, during development, is not accompanied by significant changes in SP-ergic innervation.

The caudo-cranially intermediate one-third of medullary dorsal region, the periaqueductal grey and the rostro-ventral portion of the midbrain tegmentum of adult chickens were studied in detail by means of the PAP-DAB procedure, to define further the main morphological features of the neuronal populations that in previous studies had shown VIP (Vasoactive Intestinal Polypeptide),-Somatostatin (SRIF)-, and Bombesin-like immunoreactivities. In the medulla, VIP-like immunoreactivity was detected within neuronal bodies and processes and extended down to the cervical spinal cord. SRIF-like immunoreactivity was seen only within nerve cell processes, at least a part of which could be sensitive fibre terminals. Bombesin-like immunoreactivity was observed only within neuronal processes. In the periaqueductal grey, all 3 immunoreactivities were detected within perikarya and neuronal processes, with a higher density cranially. In the rostro-ventral portion of the midbrain tegmentum, VIP-like and Bombesin-like immunoreactivities were detected (the latter being located somewhat more cranially) both in neuronal bodies and in processes. SRIF-like immunoreactivity was found in this region only in long neuronal processes.

X-ray spectra were recorded from 400-700 nm matrix areas of 0.5 micron sections prepared from the articular cartilages of 15- and 23-year-old human cadavers. The X-ray microanalysis was carried out (i) on untreated material; (ii) after removing sulfate group by a methylation procedure; (iii) after staining with a copper containing cationic phatolcyanin dye, alcian blue 8GX, preceded by carboxymethylation. K alpha peaks of sulphur could be detected in methylated (i.e. desulfated) samples. These peaks probably indicated the presence of sulphur-containing amino acids in different matrix proteins. Consequently, the measurements of sulphur despite its general use cannot be recommended for the X-ray microanalysis of sulfated glycosaminoglycans of cartilage matrix. K alpha peaks of copper could be identified after carboxymethylation and staining with alcian blue. After carboxymethylation, alcian blue can only be bound to the dissociated sulfate groups of glycosaminoglycans in the cartilage matrix. According to our spectrophotometric studies, approximately one molecule of alcian blue combined with one sulfate group. These data suggested that this technique could be used for semiquantitative estimation of sulfated glycosaminoglycans in small areas of the cartilage matrix. Using this method, we found a higher occurrence of sulfated glycosaminoglycans in the territorial matrix than in the interterritorial matrix of the intermediate and deep zones of the human articular cartilage.

Localization and development of chick heart peptidergic innervation (Substance P, VIP and Somatostatin) were investigated by means of immunofluorescence technique. The peptidergic component of the heart innervation was observed, for the first time, in older than 11 day chick embryos, i.e., subsequently to the appearance of the cholinergic component. The peptidergic structures achieve nearly full development in about 16-17 day embryos. Substance P is the most represented of the three peptides. It is localized both in nerve bundle fibers and in isolated fibers within the myocardium, the pericardium, the vessel walls; it is also present in fibers of some heart base ganglia. VIP is mostly contained in some thick single fibers travelling along the vessel walls of the heart base, the myocardium and the pericardium. Some VIP immunoreactive cells were also observed in the base ganglia. Somatostatin is mostly contained in some ganglia cells, whilst thin Somatostatin-immunoreactive fibers form a rich plexus among the atrial and ventricular myofibers, without contacting the vessel walls.

In many birds reproduction is triggered by long daylengths but, paradoxically, continued exposure to long days leads to photorefractoriness and a complete shut down of the reproductive system. As these effects are thought to be mediated through the secretion of LH-RH, immunocytochemical techniques were used to investigate changes in the LH-RH system when European starlings were exposed to different photoperiods. Starlings exposed to 11L:13D and with mature testes show strong immunostaining both of LH-RH perikarya and fibers. Photosensitive short-day (8L:16D) starlings with undeveloped testes show an almost identical distribution of strongly immunoreactive perikarya but with less dense fibre staining. However, long-day (18L:6D) photorefractory starlings with fully regressed testes, show a profound reduction in LH-RH immunostaining. Perikarya have the same distribution but show a much reduced intensity of staining and fibers had almost entirely disappeared from all regions of the brain. Preliminary observations on the ultrastructure of immunocytochemically identified LH-RH neurones are also reported.

Substance P-immunoreactive neurons were demonstrated in chick embryonic and adult trigeminal ganglion and jugular-superior ganglionic complex using FITC-immunohistochemical methods. Both small-size and large ganglion cells exhibited SP immunoreactivity, without apparent changes during embryonic and post-hatching development. SP-positive fibers could be detected in a good number in the sympathetic cranial cervical ganglion, either during embryonic development or in adult chick. No immunoreactive perikarya were observed in this ganglion. In the ciliary ganglion, both choroidal and ciliary neurons were SP-negative, whereas SP immunoreactive fibers surrounded the perikarya of both cell populations.

We investigated the presence and distribution of five glycosidic residues at the level of the endocervical columnar epithelial cells during the various phases of the menstrual cycle in healthy subjects. We utilized the lectins Wheat Germ Agglutinin (WGA), Peanut Agglutinin (PNA), Soybean Agglutinin (SBA), Concanavalin A (ConA) conjugated with colloidal gold particles as ultrastructural markers in Transmission Electron Microscopy. This technique proved to be particularly suitable for detecting glycosidic residues at the cellular level, where the mucus is produced. All the lectin-binding sites (except ConA binding sites which were not detected) appeared to be produced in significantly different amount during the cycle, supporting the hypothesis that the cyclic-related changes of the cervical mucus viscoelasticity is due to glycoconjugate cyclic variations.