By a modified cytochemical staining procedure, Mg⁺⁺- and Ca⁺⁺-ATPase were clearly localized on the surface of the limiting membrane of peroxisomes in hepatocytes of the rat. The matrix and core structure (crystalloid) were devoid of the ATPase reaction. The reaction on the peroxisomal membrane was abolished by an inhibitor of Mg⁺⁺-ATPase, 10mM p-chloromercuric benzoate (PCMB) and by the omission of the substrate, ATP-2Na, or by replacement of it with β-glycerophosphate. The reaction was not suppressed by the inhibitor of a alkaline phosphatase, 2.5mM levamisole; by an inhibitor of Na-K-ATPase, 10mM ouabain. After replacement of MgSO₄ by CaCl₂, Ca-ATPase was localized at exactly the same site where Mg-ATPase was detected.

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Histochemical localization of 5′-nucleotidase (5′-Nase) in the lymphatic endothelium of rat stomach was studied by combined light and electron microscopy. Lymphatic capillaries and blood capillaries were distinguished histochemically on cryostat sections using 5′-Nase-alkaline phosphatase double staining.
The distribution of lead-demonstrated 5′-Nase activity in the lymphatic capillaries could be determined by comparing the images of the same section as seen by light and backscattered imaging scanning electron microscopy. The specificity of the 5′-Nase reaction was obtained by inhibiting nonspecific alkaline phosphatase by including L-tetramisole in the incubation medium for 5′-Nase activity. The intensity and localization of 5′-Nase activity on the walls of lymphatic capillaries were also determined by the lead-based or cerium-based method. The intense reaction products of 5′-Nase activity were predominantly deposited on the outer surface of the plasma membrane of the lymphatic endothelial cells. The cerium-based method revealed fine and linear reaction products on the plasma membrane.

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Two methods were used to distinguish between lymphatic capillaries and blood capillaries at the light microscopic level: in one toluidine blue sections were embedded in epoxy resin following arterial perfusion-fixation, and in the other an immunohistochemical technique used basement membrane antisera (laminin, type IV collagen and fibronectin antisera) in indirect immunoperoxidase and immunogold-silver staining (IGSS) methods. The precise immunolocation of these basement membrane components around these capillaries was examined electron microscopically by pre-embedding immunoperoxidase and post-embedding immunogold techniques. The toluidine blue sections showed two types of capillaries: one with blue-stained lumens and the other with vacant lumens. Conventional electron microscopy revealed that capillaries with blue-stained lumens are lymphatic capillaries and capillaries with vacant lumens are blood capillaries by their fine structural characteristics. Immunohistochemical techniques using laminin or type IV collagen antiserum in both the immunoperoxidase and IGSS methods showed blood capillaries with continuous and linear reactions and lymphatic capillaries with absent or weak reactions beneath the endothelium. Immunostaining of fibronectin antiserum was observed nonspecifically around both types of capillaries and in connective tissue. Fine granular products in the IGSS method were in sharper contrast against the background and were more intensely stained and thinner than the DAB reaction products demonstrated by the immunoperoxidase method. Immunoelectron microscopy revealed greater immunoreactivity of laminin and type IV collagen antisera in the lamina densa around blood capillaries than in that around lymphatic capillaries. A striking feature was the presence of fibronectin antiserum in the intracellular organelles of blood capillary endothelium and on the surface of fibroblasts. In conclusion, toluidine blue staining following arterial perfusion-fixation is a useful technique for experimental pathologists, and the IGSS method using laminin and type IV collagen antisera is a practical light microscopic method of distinguishing between lymphatic capillaries and blood capillaries.

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The functional relationships between sympathetic nerve fibres and meningeal mast cells were studied in whole mounts of rat dura mater and pia-arachnoid membrane using formaldehyde histofluorescence technique. Mast cells displaying a yellow fluorescence indicative of their 5-hydroxytryptamine content were found in the dura mater, primarily perivascularly and in lesser amounts in the meningeal tissues proper without any apparent relationship with blood vessels. Bilateral removal of the superior cervical ganglion produced the degranulation of part of dura mater mast cells as well as a remarkable reduction of the intensity of their 5-hydroxytryptamine cytoplasmic fluorescence. On the contrary, the electrical stimulation of the superior cervical ganglion increased the intensity of cytoplasmic fluorescence of this kind of cells.
Taken together the above data seem to be strongly suggestive of a functional link between meningeal mast cells and sympathetic nerve fibres originating from superior cervical ganglion.

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Four experiments were carried out to investigate physical developers containing bromohydroquinone.
In experiment 1, the effect of citric acid concentrations in physical developers upon the intensity of immunohistochemical reactions was investigated, using a protein A-gold silver technique for detection of insulin. It was noted that the reactions were decreased in intensity with increasing concentrations (300, 400, 500 and 700mg/61ml) of citric acid involved. Addition of 250mg/61ml sodium citrate to the developer containing 700mg/61ml citric acid elevated pH to a value nearly comparable to that of the developer containing 300mg/61ml citric acid. Such a maneuver increased the intensity of the reaction, however, not to a level reached when the developer containing 300mg/61ml citric acid was used. Thus, it is considered that citric acid controls the developing capacity not only by modulating pH values but by complexing with silver. In experiment 2, the possibility was substantiated that the citric acid involved can be replaced with maleic acid. In experiments 1 and 2, we newly prescribed two refined physical developers (Developers 2 and 3). Developer 2 contains a larger amount (500mg/61ml) of citric acid as compared with Developer 1 which we have thus far employed primarily. In Developer 3, maleic acid is contained instead of citric acid. In experiment 3, these revised physical developers were used in protein A-gold silver or immunogold silver staining for detection of a series of antigenic substances, and proved to be excellent, in terms of the intensity, sensitivity and specificity of histochemical reaction. Further, experiment 4 in which Danscher's physical developer was tentatively used in immunostaining substantiated the superiority of the present revised developers to the one elaborated by Danscher (7).

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Comparing galanin and GAD immunoreactive axons in the rostral part of the basal forebrain complex, striking differences were observed regarding their distribution patterns and axonal morphology. In regions with very dense GABAergic innervation, galanin positive fibres were absent. The functional significance of these differences is not yet known. “Stalked” boutons were only formed by galanin containing axons, contrary to GABAergic axons which were endowed with boutons “en passage”, only. In galanin immunoreactive sections counterstained with cresylviolet most of boutons were found to be distributed in the neuropil.

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The distribution of subunit A of blood coagulation factor XIII (FXIIIa) was investigated in human pulmonary tissues by the avidin-biotin-peroxidase complex (ABC) method. FXIIIa was detected in certain connective tissue cells, including dust-laden cells of the subpleural areas and alveolar septa as well as in alveolar macrophages, but not in type I or type II alveolar epithelial cells. These findings agree well with the widely accepted conception that pulmonary macrophages are part of the general mononuclear phagocyte system (MPS). The biological role of the FXIIIa-positive pulmonary cells is also discussed in this paper.

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