A routine method for contrasting elastin at the ultrastructural level

A reliable, simple, and inexpensive method for ultrastructural investigation of elastin is described. This method uses uranyl acetate dissolved in absolute methanol, followed by an optional lead citrate counterstain. The procedure was tested on a number of animal and human tissues that had been fixed and processed differently.     

Differentiating cardiac elastin,collagen and microfibrils with NaOH at the ultrastructural level

Summary NaOH solutions extract elastin and collagen from epoxy-embedded thin sections containing rat cardiac connective tissue. Extraction results in a reverse staining effect of clastin and collagen ultrastructure. Microfibril contrast is enhanced by NaOH treatment. This phenomenon finds application in the possibility of differentiating elastin, collagen, and microfibrils at the ultrastructural level.     

A novel procedure for pre-embedding double immunogold-silver labeling at the ultrastructural level.

Pre-embedding double immunogold-silver labeling using two ultrasmall gold conjugates has not been attempted previously because a means of distinguishing labels by conjugates of identical sizes was lacking. This study investigated the feasibility of creating a particle size segregation between two ultrasmall gold conjugates through sequential immunogold incubations and silver enhancements. Two primary antibodies, mouse anti-synaptophysin and rabbit anti-glial fibrillary acidic protein (GFAP), were used in the model system. Differentiation of the double labeling was achieved by incubating with one ultrasmall gold conjugate, followed by silver enhancement, and then incubating with the second ultrasmall gold conjugate, followed by additional silver enhancement. This resulted in two groups of silver-enhanced particles: smaller particles enhanced once and larger particles enhanced twice. Electron microscopic examination revealed two readily distinguished populations of gold-silver particles within the appropriate structures, with very little size overlap. The quality of the ultrastructure permitted identification of most subcellular organelles. This procedure provides for the first time a pre-embedding immunogold-silver labeling protocol that allows the precise subcellular co-localization of multiple antigens.     

Morphology of the Turbellaria at the ultrastructural level

The paper reviews the following systems: body wall, nervous system, protonephridia, parenchyma, digestive system, reproductive system, and includes a summary of the literature. New information is presented for the catenulid and neoophoran body wall-construction, the kalyptorhynch proboscis, the catenulid and haplopharyngid protonephridial construction, and the prolecithophoran spermatozoon and female germ cell. Examples of new features, as well as examples of how electron microscopy has clarified the relative position of structures and their substructures are given from the subcellular level to the organization of whole organs. Fine structural features linking different turbellarian orders are summarized. They apparently support Karling's (1974) latest assessment of the affinities between the turbellarian orders which is based primarily on light histological data, they add the recognition of a special link between the Macrostomida and Haplopharyngida and they suggest the existence of three main evolutionary lines within the Turbellaria.     

Indirect hemagglutination test at the ultrastructural level]

A study on ultra-thin sections was made of the preparations of agglutinate produced during the reaction of the immunoglobulin erythrocytic diagnostic agent with dry corpuscular Rickettsia prowazeki antigen, fluoresceine isothiocyanate labeled, and also SRBC used for the preparation of the diagnostic agent after formalinization, tannin treatment, sensitization with hyperimmune horse serum immunoglobulins and lyophilization, respectively. Formalin and tannin treatment of erythrocytes failed to be reflected on the ultrastructure of their cellular membranes; the treatment with hemosensitin was accompanied by the appearance of spheroid protrusions of the erythrocyte cytoplasmic membrane with the preservation of its three-layer structure. Specific interaction of sensitized erythrocytes with the antigen corpuscles was expressed morphologically in their apposition or connection through a gap of 20--30 nm.     

Nile blue A as a fluorescent stain for poly-beta-hydroxybutyrate.

Poly-beta-hydroxybutyrate granules exhibited a strong orange fluorescence when stained with Nile blue A. Heat-fixed cells were treated with 1% Nile blue A for 10 min and were observed at an excitation wavelength of 460 nm. Glycogen and polyphosphate did not stain. Nile blue A appears to be a more specific stain for poly-beta-hydroxybutyrate than Sudan black B.     

Pt-staining of peroxidatic reaction products at the ultrastructural level

The use of H2PtCl6 is proposed for the selective visualization of the poly-DAB reaction product created, in aldehyde-fixed tissue, with the cytochemical reaction according to Graham and Karnovsky (1966) or to Hoefsmit (1975). At sites known to contain peroxidatic activity, at the ultrastructural level, an electron-dense reaction product is acquired in otherwise unstained ultrathin sections. The presence of the element platinum in these sites has been demonstrated by X-ray microanalysis, for both the endogenous peroxidase and peroxidase conjugated to antibodies. The absolute platinum concentration has been established in erythrocytes and the granules in eosinophils and monocytes by co-embedded, Pt-containing Chelex ion-exchange beads next to the cells. By the application of the method of integrated morphometrical and chemical analysis (de Bruijn and Zeelen 1984; de Bruijn 1985; de Bruijn and Cleton 1985), both the elemental concentration and the area occupied have been calculated for eosinophil granules. The mean Pt net-intensity values of the cytoplasmic areas, known not to contain the enzyme peroxidase has been measured, and compared to the mean net-intensity Pt values of the granules. It was noted that the cytoplasmic Pt net-intensity values were not zero. The two sets of values are expressed as a mean Pt granule/cytoplasm ratio, this ratio creates a value for the "selectivity" of the reaction. The application of a postfixation reaction with OsO4- containing media, at pH 7.4, in addition to the H2PtCl6 reaction, resulted in a contrasted poly-DAB reaction product at all sites known to contain peroxidatic activity. However, X-ray microanalysis revealed that in addition to platinum, osmium was present.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pt-Staining of peroxidatic reaction products at the ultrastructural level

Summary The use of H₂PtCl₆ is proposed for the selective visualization of the poly-DAB reaction product created, in aldehyde-fixed tissue, with the cytochemical reaction according to Graham and Karnovsky (1966) or to Hoefsmit (1975).At sites known to contain peroxidatic activity, at the ultrastructural level, an electron-dense reaction product is acquired in otherwise unstained ultrathin sections. The presence of the element platinum in these sites has been demonstrated by X-ray microanalysis, for both the endogenous peroxidase and peroxidase conjugated to antibodies.The absolute platinum concentration has been established in erythrocytes and the granules in eosinophils and monocytes by co-embedded, Pt-containing Chelex ion-exchange beads next to the cells.By the application of the method of integrated morphometrical and chemical analysis (de Bruijn and Zeelen 1984; de Bruijn 1985; de Bruijn and Cleton 1985), both the elemental concentration and the area occupied have been calculated for eosinophil granules. The mean Pt net-intensity values of the cytoplasmic areas, known not to contain the enzyme peroxidase has been measured, and compared to the mean net-intensity Pt values of the granules. It was noted that the cytoplasmic Pt net-intensity values were not zero. The two sets of values are expressed as a mean Pt granule/cytoplasm ratio, this ratio creates a value for the selectivity of the reaction.The application of a postfixation reaction with OsO₄-containing media, at pH 7.4, in addition to the H₂PtCl₆ reaction, resulted in a contrasted poly-DAB reaction product at all sites known to contain peroxidatic activity. However, X-ray microanalysis revealed that in addition to platinum, osmium was present.A reaction mechanism for the cytochemical poly-DAB contrast-staining at low pH, based upon the reaction proposed by Wild (1963), is postulated.In honour of Prof. P. van Duijn     

Lead-haematoxylin as a stain for endocrine cells

Summary A modification of MacConaill's lead-haematoxylin has been found to stain several endocrine cells producing polypeptides and monoamines, particularly A and D cells of the pancreatic islet, thyroid C cells, gastro-intestinal enterochromaffin cells, gastric G and X cells, pituitary ACTH and MSH cells, adrenal medullary cells, and chemoreceptive cells of the carotid body. A careful comparison of the results of this method with those of HCI-basic dye method and of monoamine methods suggested that carboxyl groups of proteins may be the main binding site of lead-haematoxylin. Experiments with various pretreatments of tissue sections support such a hypothesis. The possibility that biogenic amines take also some part in the staining cannot be ruled out.     

Development of a simple embedding procedure allowing immunocytochemical localization at the ultrastructural level

Immunobed solution A is a water-soluble acrylic compound recently developed for immunocytochemical localization at the light microscopic level. In this study, we combined it with methyl methacrylate (MMA) to achieve sufficient hardness to obtain ultra-thin sections. Samples of platelets were dehydrated and embedded in the water-soluble acrylic mixture (WSAM). The embedding process was carried out at 4 degrees C and final polymerization was induced with either chemical (benzoyl peroxide) or physical (UV light) catalysts. Tubulin was localized at the ultrastructural level in sections embedded according to these two methods. Results were compared with those obtained in platelets processed in Lowicryl. Dehydration and embedding with the WSAM yielded a preservation of antigenicity similar to that obtained in Lowicryl. The new procedure benefits from the low temperature achieved during polymerization, providing good ultrastructural morphology and immunolocalization of protein antigens with the simplicity of a routine embedding procedure for light microscopy.