An applicable method of cryofixation to immunocytochemistry was examined. Fresh tissue blocks of rat pancreas and parotid gland were quickly frozen by the metal contact method using liquid helium and freeze-substituted with one of the following media kept at -80°C for 36hr; pure acetone, 0.1% glutaraldehyde (anhydrous) in acetone, approximately 0.2% paraformaldehyde in acetone, and 10% acrolein in acetone. After freeze-substitution fixation, tissue blocks were embedded in Araldite mixture. Thin sections mounted on nickel grids were processed for immunocytochemical localization of amylase according to the multiple-step protocol of the protein A-gold immunostaining method by Bendayan and Duhr (4). They were then postfixed with 2.5% glutaraldehyde in PBS and stained with uranyl acetate and lead citrate for electron microscopy. Good results were obtained from the materials substituted with glutaraldehyde or paraformaldehyde in acetone. The ultrastructural features of the cells were preserved well, similar to those in the materials substituted with OsO4 in acetone except for negative images of the membranous structures. Secretory granules, condensing vacuoles, and Golgi cisterns were labeled well with immunogold. Labeling density was much higher in the present materials than in those processed by conventional chemical fixation, and the intensity of labeling increased in proportion to increasing electron density of the materials contained within individual subcellular compartments. These results indicate that an application of the cryofixation method is useful for improving resolution and specificity in immunocytochemical postembedding staining.
Ultrastructural localization of the different types of phosphatase activities (Ca++ activated ATPase (Ca++-ATPase), Mg++ activated ATPase (Mg++-ATPase), Alkaline phosphatase (AlPase)) in the normal minor salivary glands of adult human lower lip were studied by the enzyme histochemical method. Intense reaction products resulting from Ca++-ATPase activity were observed on adjoining plasma membrane of the acinar, the intercalated ductal and the myoepithelial cells, however little reaction product was found on the luminal or basal plasma membrane of these cells. Localization pattern of Mg++-ATPase activity was almost equal to that of Ca++-ATPase activity. AlPase activity was demonstrated on the luminal and lateral surfaces of endothelial cells and on the inside substrate of the capillary, while no reaction product was present on acinar, intercalated ductal or myoepithelial cells. These observations are discussed with respect to their physiological significance.
Granules of the secretory cells of guinea pig middle ear mucosa, including those of the eustachian tube, were studied by electron microscopic lectin-gold complex postembedding staining. Electron-lucent (EL) granules were intensely stained with wheat germ agglutinin (WGA)-gold and Helix pomatia agglutinin (HPA)-gold. Within these granules WGA-gold stained the entire electron-dense core and the surrounding filamentous structure. In contrast HPA-gold stained the filamentous structure and only the lesser electron-dense portion of the core. The electrondense (ED) granules were uniformly labeled with WGA, but did not stain with HPA. In intermediate cells, a small number of a third type of granule (intermediately electron-dense (ID)), which resembled ED granules but were less electron-dense and had a peripherally electron-lucent zone adjacent to the limiting membrane, showed a weak reactivity with HPA only on the limiting membrane. These results suggested that one type of granule could transform into another.
With a view to improving the immunohistochemical technique for the demonstration of bioactive substances, i.e., neuropeptides and biogenic amines in the mammalian central nervous system, the procedures of immunoperoxidase methods were examined and some modifications for obtaining consistent results were developed. Brains of pharmacologically untreated animals were utilized as the material of the present study. The time through thoracotomy and preperfusion was reduced as much as possible. Perfusion fixation was performed at an increased rate, using a blood pump (hemolizer). The first fixative was a mixture of 4% formaldehyde, 0.2% picric acid, and 0.5% glutaraldehyde in phosphate buffer; the second was 4% formaldehyde and 0.2% picric acid in phosphate buffer acidified to pH6.5 with acetic acid; and the third was buffered formaldehyde solution. The osmotic pressure of all these fixatives was 1550-1650mOsM. Sections 25μm thick were produced on a Microslicer, followed by application of the freezing-thawing technique. The free-floating sections were incubated in a low concentration of antibody solution diluted by 0.5% Triton X-100 in phosphate buffer for a longer period than usual, under cool condition. The reactive substances resulting from the avidinbiotin-peroxidase complex method were enhanced by osmication. With these methods, the serotonin and GRH neurons could be clearly and finely visualized.
The histo- and cytochemical localization of guanylate cyclase activity was examined in the newborn rat retina during the first three postnatal weeks. The reaction was first observed on the 3rd postnatal day on the apical plasmalemma of the retinal pigment epithelium. The reaction was seen on the disk and plasmalemma of the premature short outer segments from the 10th day. From the 14th day, the enzyme localization seemed to be the same as shown in the adult rat. The reaction products were restricted to the apical plasmalemma of the retinal pigment epithelium and disk and plasmalemma of the tip of the outer segments.
In a series of mammalian tissues, the reactions of cuprolinic blue with glycoconjugates were studied under light microscopy. The dye solutions were adjusted to pHs 1.0, 1.5, 2.5, 3.0 and 5.6 and applied to different glycoconjugate-containing tissues in combination with certain chemical modification and enzyme digestion procedures. The results obtained revealed that (a) the ortho- and metachromatic staining intensities with the dye increase with advancing pH levels, (b) most proteoglycan-containing structures exhibit purple shades of metachromatic stainings at higher pHs, whereas glycoprotein-containing ones show reddish shades of staining at every pH level and (c) the effects of chemical modification and enzyme digestion procedures upon the staining reactions lead to a possible correlation of individual glycoconjugates with their cuprolinic blue stainabilities in tissues.
Saltatory intracellular lysosomal movements were enhanced by both fluid-phase pinocytosis (horseradish peroxidase; HRP) and adsorptive pinocytosis (peroxidase anti-peroxidase; PAP) in cultured rat alveolar macrophages. To elucidate the role of cytoskeletal elements in the regulatory mechanism of lysosomal movements related to the autophagy and heterophagy, the effects of actin filament destabilizers (cytochalasin B, D) and antimicrotubular drugs (colchicine, nocodazole) on the lysosomal movement induced adsorptive pinocytosis of PAP were investigated by cytochemical electron microscopy. In the cultured alveolar macrophages, the pinocytosis of PAP promoted lysosomal movements and the extension of nematolysosomes (thread-like lysosomes) within the cytoplasm. The actin filament destabilizers inhibited these lysosomal movements and forming process of nematolysosomes, while the drugs had little or no inhibitory effect on pinocytosis of PAP. The antimicrotubular drugs also had an inhibitory effect on the appearance of nematolysosomes. Moreover, these drugs led to another type of lysosomal transformation, i.e., wrapping lysosomes which are thought to be one process of autophagy. The present study using actin filament destabilizers and antimicrotubular drugs clarifies two distinct types of lysosomal transformations, which are nematolysosomes and wrapping lysosomes. It is suggested that cytoskeletal elements play an important role in regulating the movement and transformation of lysosomes in the macrophages.
In the previous report, we showed that lysosomal movements and transformations were greatly affected by actin filament destabilizers and antimicrotubular drugs (3). To confirm this circumstantial evidence suggesting an important role of cytoskeletal elements in the regulation of lysosomal movements, the direct morphological interaction of actin filaments with lysosomes in rat alveolar macrophages were examined by several electron microscopic techniques. In the observation of ultrathin sections of alveolar macrophages, some filamentous elements appeared to be closely associated with lysosomes in the cytoplasm. The three-dimensional observations in the whole mount macrophages and the cells cleaved by the dry-blowing method also demonstrated that the filamentous elements were in contact with lysosomes in situ. The organization of filamentous elements closely associated with lysosomes was mainly composed of actin filaments which could be decorated with heavy meromyosin (HMM), although a few intermediate filaments existed among them. In a cell-free experimentation in vitro, when G-actin was mixed with the lysosomal fraction isolated from alveolar macrophages and then polymerized to Factin, it was found that F-actin filaments appeared to be in contact with the membrane of lysosomes by negative-stained electron microscopy. In vitro interaction of actin filaments with isolated lysosomes implies that the lysosomal membrane has the ability to bind to actin filaments. This finding of in vitro experimentation coincides with those of in situ observation. These results provide substantial evidence for the interaction of actin filaments with the lysosomal membrane, which suggest that actin filaments participate in regulating intracellular lysosomal movements.