Ca++-activated adenosine triphosphatase (Ca++-ATPase) activity in the epithelial cells of guinea pig ciliary body was demonstrated histo- and cytochemically with the use of a newly developed method (Ando et al., 1981). In nonpigment ciliary epithelial cells fixed for 60min in paraformaldehyde, intense reaction was observed on basolateral plasmalemma and mitochondria. In contrast, an intense reaction was localized only on mitochondria and gap junctions but not on the plasmalemma in the pigment epithelial cells. From the controls, this reaction was completely Ca++- and ATP-dependent. The activity in fixed tissue with a mixture of paraformaldehyde and glutaraldehyde still remained moderately on the plasmalemma, but was completely abolished on mitochondria and gap junctions. The activity in the tissue after incubation in the medium with 2mM or 3mM Ca++ concentration remained on the plasmalemma but almost disappeared from the mitochondria and gap junctions. Utilization of 1mM or lower Ca++ concentration failed to show enzyme activity. When tissue was incubated in the medium containing both 5 or 10mM Ca++ and Mg++, the reaction products on the mitochondria were still intense. However, the activity on the plasmalemma was markedly weakened. When Mg++ were used instead of Ca++, a distinct reaction was also found on mitochondria, but not on the plasmalemma. Our results clearly revealed that Ca++-ATPase activity was localized on the plasmalemma, mitochondria and gap junctions in the ciliary epithelium. It is noteworthy that a much more intense reaction existed on the basolateral plasmalemma which has been suggested to play an important role for aqueous humour secretion facing the posterior chamber in the nonpigment epithelium.
IgG-F(ab′)2 from normal human serum was incubated and bound to paraformaldehyde fixed bacteria of K. pneumoniae and E. coli. The IgG-F(ab′)2 that bound to the bacteria was each solubilized from the bacteria by glycine-HCl buffer, pH 2.5, and was again reacted with the paraformaldehyde fixed bacteria. It was found that the IgG-F(ab′)2 solubilized from E. coil and K. pneumoniae each showed binding to respective bacteria but not to unrelated bacteria, these findings indicating that the binding of the IgG-F(ab′)2 with bacteria is, if not all, through specific antigen-antibody reaction.
Immunohistochemical staining with keratin antiserum was performed on formalin-fixed paraffin sections of 63 specimens of human salivary gland tumors including normal salivary glands. Strong keratin staining was evident in ductal segments of the parotid and submandibular glands. Significantly intense staining for keratin proteins was found in luminal epithelia of tubular, duct-like, and cystic dilated epithelial structures in pleomorphic adenoma, squamous metaplastic regions and squamous carcinomas. Myoepithelial cells in normal salivary glands were not clearly positive for keratin staining, nor were tumor cells which were possibly myoepithelium-derived, whereas, modified myoepithelial tumor cells in hyalinous and/or myxomatous regions displayed varying degrees of keratin staining. Epithelial cells of Warthin's tumor were strongly reactive. Mucoepidermoid carcinomas also showed positive keratin staining in proportion to the number of mixed epidermoid cells present. Adenoid cystic carcinomas showed slight staining for keratin in scattered tumor cells.
The binding of horseradish peroxidase (HRP)-conjugated lectin was studied in the oral mucosa of several mammals. The following lectins were used; Con A, PNA, SBA, DBA, RCA-1, UEA-1, and WGA. Con A staining was exhibited strongly in the connective tissue cells, moderately in germinal cells, and negatively in the keratinized layer. However, following periodate oxidation (PA/Con A) it revealed a different distribution patterns as compared to single Con A staining; i.e., PA/Con A was limited to the spinous cell epithelium. Lectin binding gave weak results in basal cells, strong in spinous cells, and showed no effect in keratinized layers. Using lectins-PNA, SBA, RCA-1, UEA-1, and WGA-, the effects in the oral squamous epithelium revealed zonal or regional distribution in their respective individual layers except for a few exceptions.
Intramitochondrial distribution of three acyl-CoA dehydrogenases in parenchymal cells of rat liver was investigated by means of a post-embedding immunocytochemical technique. Vibratome sections of rat liver fixed with 4% formaldehyde+1% glutaraldehyde were embedded in Lowicryl K4M at -20°C. Ultrathin sections were incubated with antibodies against short-chain acyl-CoA dehydrogenase (SCAD), medium-chain acyl-CoA dehydrogenase (MCAD), and long-chain acyl-CoA dehydrogenase (LOAD), respectively, followed by a protein A-gold complex. The gold particles representing the antigenic sites for three acyl-CoA dehydrogenases were present exclusively in mitochondria. Quantitative analysis of labeling density confirmed that the labeling density for the enzymes was significantly high in the mitochondria. The histogram analyzing the distribution pattern demonstrated that the most of the gold particles were found in zone with a width of approximately 12nm along the innermembrane. The results suggested that mitochondrial acyl-CoA dehydrogenases are associated with the mitochondrial innermembrane.
In the present study the effects of contaminating proteinase activities in commercially available preparations of chondroitinases and keratanase were tested upon the histochemical results obtained by their digestion procedures. Tissue sections from the ear and tracheal cartilages, aorta, umbilical cord and cornea of mammals were subjected to staining procedures after prior digestion with the enzyme solutions with or without proteinase inhibitors. In a substantial number of tissue sections examined, the addition of proteinase inhibitors to the enzyme solutions appeared to suppress the decline in intensity of the staining reaction induced by the enzyme digestions. These results are taken to indicate that the commercially available preparations of chondroitinases and keratanase contain significant amounts of contaminating proteinases. When these enzyme preparations are employed as histochemical reagents the addition of proteinase inhibitors to the enzyme solutions is indispensable for precise localization of the enzyme substrates in tissues.
We investigated the expression patterns of (1) intermediate filament proteins in the developing CNS according to the immunohistochemical and immunoblotting techniques and (2) glycosyl residues exposed on the cell membranes in the fetal brain using histochemical techniques. In the early neural tube which was composed of matrix cells (stage I), vimentin was found predominantly in the basal processes of these cells. After the beginning of neuron production (stage II), vimentin was present in “radial fibers”, guideropes for migrating neuroblasts, which could be regarded as elongated basal processes of matrix cells. In this stage of neurogenesis, neurofilament protein was detected in the processes running parallel to radial fibers. These processes seemed to be those of migrating neuroblasts. Glial fibrillary acidic protein was detected for the first time after the cessation of neuron production (stage III). These results indicate the absence of glial cells in the early stages of neurogenesis (stages I and II). The lectin-histochemistry of the fetal brain implied the presence of abundant terminal sialyl and N-acetylglucosaminyl residues on the plasma membranes of various cells and fibers. On the other hand, it was suggested that terminal galactosyl residues on radial fibers are much fewer in the corticogenetic region than in the non-corticogenetic place. This difference in the amount of terminal galactosyl residues could be related to the mechanism of corticogenesis. A possible role of galactosyltransferase in this mechanism was suspected.
Using new methods which perform 3H-thymidine autoradiography and AChE histochemistry on the same sections, the interrelationship among loss of proliferating ability, morphological development and increase of AChE activity during the course of differentiation of the neural tube cells were investigated in early chick embryos. The neural tube wall consisted of four kinds of cells: (1) AChE negative spindle shaped cells, (2) weakly positive spindle shaped cells showing AChE activity in the cisternae of the nuclear envelope and in a few short profiles of r-ER, (3) moderately positive spindle shaped cells showing AChE activity in the cisternae of the nuclear envelope and in a moderate number of r-ER profiles and (4) intensely positive large round cells showing AChE activity in the cisternae of the nuclear envelope and in a large number of r-ER profiles. Nuclei of the former three kinds of cells slet located in the ependymal layer, while the AChE intensely positive cells were in the mantle layer. The AChE negative and weakly positive cells were capable of proliferation and were regarded as undifferentiated neuroepithelial cells. The AChE moderately positive and intensely positive cells were, in contrast, no longer capable of proliferation and were regarded as neurons, especially the former as newly formed neurons. These findings indicate that the r-ER increases rapidly in number and in volume in newly formed neurons, soon after their final cell division, and that AChE activity increases in the neurons parallel to the development of the r-ER.
Ontogeny of various kinds of neuropeptides such as cholecystokinin-8-like (CCKI), leucine-enkephalin-like (ENKI), neurotensin-like (NTI), substance P-like (SPI) and somatostatin-like immunoreactive (SOMI) structures was examined in the rat brain using immunocytochemistry. The prenatal early appearance of immunoreactive structures in the brain is a common feature in the development of the peptidergic system. However, the developing patterns of peptides vary according to the peptides themselves, among individual peptides depending on the regions. The early appearance of peptides in the fetus brain suggests the role of peptides in the development of the brain in addition to their neuromodulator role.
A combined method of immunocytochemistry and [3H]thymidine autoradiography was devised to analyze the time of production of chemically characterized neurons. The method consisted of the following steps: 1) [3H]thymidine injection in pregnant animals, 2) immunocytochemistry using peroxidase and anti-peroxidase (PAP) techniques, and 3) autoradiography. The technical problems of these methods are discussed.
Reeler mutant mouse is characterized by the derangement of neuron positions in the cerebral cortex. In order to analyse the pathogenesis of the reeler abnormality, we have here analysed the chimera composed of reeler and normal on a half-and half basis judged from the coat color and GPI isozyme pattern. By hematoxylin-eosin staining and Golgi staining, the cerebral cortex was found to be normalized. The cortico-spinal tract neurons retrogradely labelled after injection of horseradish peroxidase into the lumbar cord were situated in layer V (the inner pyramidal layer) in the chimera, as in the normal mouse, whereas in the reeler such neurons were scattered diffusely throughout all levels of the corresponding cortical area. These observations suggest that reeler mutation does not directly affect the neurons and that abnormalities in process formation of neurons and fiber trajectory are consequential upon malposition of the cell.
Proliferation and differentiation of glial cells in mature and developing brains of rodents were investigated. Autoradiography, immuno- and enzymehistochemistry and computer graphic analysis were used in this study. The following results were obtained; In the white matter of the adult brains astroglia seem to proliferate in response to injuries, while the main proliferating glial cells in the gray matter, i.e. hippocampal gray matter, or in the facial nucleus, are microglia. Computer graphic analysis disclosed that total volume of microglial cell body in the nucleus suffered from retrograde degeneration increases up to five times of that in the normal nucleus. It was suggested that proliferating microglia transform into fibrous astroglia, and thus, they were supposed to play a significant role in the repair process of damaged brains. The results of autoradiographical studies on the developing brains indicated that immature glioblasts are directly formed from ependymoglioblasts, the progeny of the matrix cells. We could trace smooth morphological transition from glioblasts to astroglia or to microglia.