The Fine Structure of Endothelium of Large Arteries

Endothelium of large arteries from several species was studied in thin sections with the electron microscope. Before sacrifice, some animals received an intravenous injection of colloidal thorium dioxide which was visualized in the sections. Surface replicas were prepared by carbon evaporation on either frozen-dried endothelium or on endothelium dried by sublimation of naphthalene with which the tissue had been impregnated. Cell boundaries, stained with silver, were observed in sections and also from the surface by stripping off the inner part of the endothelium. In addition to the usual cytoplasmic organelles, the endothelial cells showed certain characteristic features, namely, large invaginated pockets communicating with the arterial lumen, numerous much smaller vesicular structures immediately under the plasma membrane and apparently also communicating with the lumen, and inclusions, into which injected thorium particles were incorporated. Intercellular boundaries appeared as regular double membranes in thin sections, and they were outlined by a double row of silver granules after silver staining. No evidence was obtained of permeation of intracellular spaces by colloidal thorium.     

Post-embedding double-labeling of antigen-retrieved ultrathin sections using a silver enhancement-controlled sequential immunogold (SECSI) technique.

In electron microscopy, the post-embedding immunogold technique provides a high degree of resolution and the possibility of quantitation owing to the intrinsic characteristics of the colloidal gold marker. Application of this technique to the subcellular localization of multiple antigens by differential labeling using gold markers of different sizes, or to double labeling using the same primary antibody isotype with serial silver enhancement, has been reported. We have incorporated this double labeling technique into a modified procedure that produces excellent labeling and ultrastructural preservation, even after exposure of ultrathin sections large enough to cover a 300- micro m-diameter single-hole grid to hot antigen retrieval solutions and prolonged labeling protocols.     

Ultrastructural autometallography: a method for silver amplification of catalytic metals

The autometallographic technique involves application of a silver bromide-containing emulsion on the surface of ultrathin sections placed on grids that are subsequently exposed to a photographic developer. In tissue sections from animals treated intravitally with gold, silver, or mercury compounds, accumulations of the metals are visualized by autometallography and can be used for quantitative studies. After amplification, sections can be stained with lead citrate and uranyl acetate. Using autometallography, particles of colloidal gold dispersed in a film of gelatin showed a time-dependent growth and were gradually amplified up to 3.5-fold after 15 min of development. Hence the method may prove useful tracing colloidal gold particles in sections with low particle density, and be a powerful tool for revealing metals in biological tissues.     

Investigation of immunogold-silver staining by electron microscopy

Deposition of metallic silver on colloidal gold immunoreagents has been shown to be a very sensitive immunostaining technique capable of detecting low levels of immunoreactivity in tissue sections. Using electron microscopy we have shown that immunolabelling is highest with small sizes of gold which can penetrate sections better and achieve higher densities of particles in the section than larger particles. Chemical permeabilisation of the embedding medium aids the penetration of colloidal gold. The silver enhancement step in immunogold-silver staining was shown to be progressive, allowing optimisation of staining and the selection of the final size of silver deposits required. Some poorly understood features of the technique are rationalised and the additional knowledge gained will aid the wider application of this method.     

Investigation of immunogold-silver staining by electron microscopy

Summary Deposition of metallic silver on colloidal gold immunoreagents has been shown to be a very sensitive immunostaining technique capable of detecting low levels of immunoreactivity in tissue sections. Using electron microscopy we have shown that immunolabelling is highest with small sizes of gold which can penetrate sections better and achieve higher densities of particles in the section than larger particles. Chemical permeabilisation of the embedding medium aids the penetration of colloidal gold. The silver enhancement step in immunogold-silver staining was shown to be progressive, allowing optimisation of staining and the selection of the final size of silver deposits required. Some poorly understood features of the technique are rationalised and the additional knowledge gained will aid the wider application of this method.     

Cationic colloidal gold — a probe for light- and electron-microscopic characterization of acidic glycoconjugates using poly-l -lysine gold complex

Summary Cationic colloidal gold (CCG) was used to characterize acidic glycoconjugates in semithin and ultrathin sections of rat large intestine and salivary glands embedded in hydrophilic Lowicryl K4M resin. It was prepared from poly-l-lysine and 10 nm colloidal gold solution. The staining of CCG in semithin sections was amplified after photochemical silver reaction using silver acetate as a silver ion donor and examined under bright-field and epi-illumination microscopy. CCG adjusted to various pH levels was tested on various rat tissues whose histochemical characteristics with regard to acidic glycoconjugates are well known. At pH 2.5 CCG labelled tissues containing sialylated and sulphated acidic glycoconjugates such as the apical cell surface, mucous cells in the distal and proximal colon, and acinar cells of the sublingual gland. In contrast, CCG at pH 1.0 labelled tissues containing sulphated acidic glycoconjugates such as mucous cells in the upper crypt of the proximal colon and mucous cells in the whole crypt of the distal colon. This specificity of CCG was verified by the alteration of CCG staining following several types of cytochemical pretreatment. These results were further confirmed by electron microscopy. CCG staining is thus a useful postembedding procedure for the characterization of acidic glycoconjugates at both the light- and electron-microscopic levels.     

STUDIES ON THE CORNEA : I. The Fine Structure of the Rabbit Cornea and the Uptake and Transport of Colloidal Particles by the Cornea in Vivo

Physiological studies have demonstrated that ions, as well as large molecules such as hemoglobin or fluorescein, can diffuse across and within the cornea. Most of the substrates for corneal metabolism are obtained from aqueous humor filling the anterior chamber. In order to receive its nutrients and in order to maintain its normal conditions of hydration, the avascular cornea must transport relatively large amounts of solute and solvent across the cellular layers which cover this structure. It has been suggested in the past that there may be a morphological basis for the transport of large amounts of solvents and solutes by cells by the mechanism of pinocytosis. The use of electron-opaque markers to study fluid movements at the electron microscope magnification level was described by Wissig (29). The present study describes the fine structure of the normal rabbit cornea and the pathways of transport of colloidal particles by the cornea in vivo. Rabbit corneas were exposed in vivo to suspensions of saccharated iron oxide, thorium dioxide, or ferritin by injection of the material into the anterior chamber. In other experiments thorium dioxide or saccharated iron oxide was injected into the corneal stroma, producing a small bleb. Particles presented at the aqueous humor surface of the rabbit corneal endothelium are first attached to the cell surface and then pinocytosed. It appears that the particles are carried around the terminal bar by an intracellular pathway involving the pinocytosis of the particles and their subsequent transport in vesicles to the lateral cell margin basal to the terminal bar. Particles introduced at the basal surface of the endothelium (via blebs in the corneal stroma) are apparently carried through the endothelial cells in membrane-bounded vesicles without appearing in the intercellular space. There appears to be free diffusion of these particles through Descemet's membrane and the corneal stroma. The stromal cells take up large quantities of the particles when blebs are injected into the stroma.     

Immunoelectron microscopic visualization of the transcytosis of low density lipoproteins in perfused rat arteries

A postembedding labeling technique was employed to visualize human native low density lipoproteins (LDL) during transcytosis in rat arterial endothelium. For this purpose human LDL was perfused through rat vasculature before fixation and processing for immunoelectron microscopy. The LDL particles were located on sections by anti-human apolipoprotein B-100 (LDL) antibodies and secondary antibodies or protein-A conjugated to 10-nm colloidal gold. LDL molecules were seen in plasmalemmal vesicles as well as in the subendothelial space. No colloidal gold was found in the intercellular junctions. Perfusion with reductively methylated LDL, which cannot bind to the LDL receptor, gave a similar labeling pattern, indicating that transcytosis of LDL via plasmalemmal vesicles is most likely receptor independent. Furthermore, the passage of LDL through intact vascular endothelium is a vesicular transport rather than an intercellular diffusion process.     

The coating of mouse myocardial cells. A cytochemical electron microscopical study.

The coating of mouse myocardial cells has been investigated with a variety of cytochemical methods. The coating of the surface membrane gives a positive reaction with ruthenium red, colloidal thorium, phosphotungstic acid (PTA) at low pH, silver methenamine after periodic oxidation (PA-silver technique) and with silver proteinate after periodic oxidation and thiocarbohydrazide treatment (PA-TCH-silver technique). The coating of the T system gives almost similar results. The nexuses do not react with PTA nor with the PA-silver and PA-TCH-silver techniques, but they are strongly stained with ruthenium red which reveals periodic structures in their gaps. The specificities of the colloidal thorium technique and PAT staining have been tested by chemical treatments (methylation, acetylation, saponification), enzymatic digestions (pronase, trypsin, hyaluronidase, neuraminidase) and carbohydrate extractions (with 0.1 N NaOH and 0.05 M H2SO4). These cytochemical data indicate, considering the specificity of the reactions, that the coating of the membrane surface and the T system contains polyanionic groups. A part of them, at least, would belong to a carbohydrate-containing material (glycoproteins), whereas at the level of nexuses the sugar residues would probably be absent.     

Electron histochemistry and ultrastructural localization of carbohydrate-containing substances in the sheath of Volvox.

The location and characteristics of carbohydrate-containing structures within the intact sheath of Volvox were studied by 3,3'-diaminobenzidine tetrahydrochloride-osmium, colloidal iron, colloidal thorium, ruthenium red and periodic acid-silver methenamine staining. The sheath consists of external and internal fibrillar layers separated by a tripartite structure. The external layer reacts positively with 3,3'-diaminobenzidine tetrahydrochloride, colloidal iron, colloidal thorium and ruthenium red, indicating that it contains acid mucosaccharides. Staining in the external layer is abolished by Ba(OH)2 treatment. The tripartite structure and internal fibrillar layer contain periodic acid reactive groups which do not occur in the external layer. Under certain conditions, reactions between the cationic dyes and the internal material were also observed. It is postulated that the internal matrix of the sheath contains glycoproteins or a mixture of acid mucosaccharides and glycoproteins. Possible functions of the sheath material are discussed.     

STUDIES ON THE PERMEABILITY OF LYMPHATIC CAPILLARIES

The passageway for interstitial fluids and large molecules across the connective tissue lymph interface has been investigated in dermal lymphatic capillaries in the ears of guinea pigs. Numerous endothelial cells overlap extensively at their margins and lack adhesion devices at many points. The observations suggest that these sites are free to move as a result of slight pressure changes. Immediately following interstitial injections of tracer particles (ferritin, thorium, carbon, and latex spheres), many of the overlapped endothelial cells are separated and thus passageways are provided between the interstitium and lymphatic lumen. Tracer particles also occur in plasmalemmal invaginations along both connective tissue and luminal fronts. All of the tracer particles accumulate within large autophagic-like vacuoles. Very few particles of ferritin are observed in the endothelium after 24 hr; however, the vesicles containing the nonprotein tracer particles (carbon, thorium, and latex) increase in size and content and remain within the lymphatic endothelial cells up to 6 months. The role of vesicles in the transport of large molecules and particles is discussed in relation to the accretion of tracer particles within large vesicles and autophagic-like vacuoles in the endothelial cytoplasm.     

Microdistribution of alpha particles in pathological sections of tissues from thorotrast patients detected by imaging plate autoradiography

Thorotrast is a colloidal suspension of radioactive (232)ThO(2) that naturally emits alpha particles (90%), beta particles and gamma rays (10%). Thorotrast was used as a radiographic contrast agent in the 1930s-1950s; it caused liver cancer several decades after injection because of its life-long deposition and exposure. Determination of the amount and the distribution of radioactive thorium are essential for assessment of radiation risks. We visualized alpha particles on ordinary archival tissue sections using an imaging plate and a BAS5000 image analyzer. Furthermore, we confirmed that the imaging system is sensitive enough to detect alpha particles and accurate in measuring the total amount of thorium deposited in the organ from a single tissue section. This method revealed that the amount of thorium deposited in tumor tissue is correlated to that in non-tumor tissue. Thorotrast deposition was not associated with DNA damage determined by histochemistry. In combination with histological findings, it is suggested that radioactive thorium always migrates within the deposited organs by macrophages, and that the organs are evenly exposed to alpha particles.     

Immunolabeling of grapevine flavescence dorée MLO in salivary glands of Euscelidius variegatus: a light and electron microscopy study

Flavescence dorée (FD), a grapevine yellows disease, is caused by a mycoplasma-like organism (MLO). A colloidal gold indirect immunolabeling technique identified MLO in salivary glands of a vector leafhopper, Euscelidius variegatus. After aldehyde fixation, tissue samples were prepared by cryoultramicrotomy or embedding in acrylic resins. Double fixation with aldehydes and osmium retroxide, followed by embedding in epon, was also performed. Thin or semi-thin serial sections were treated with polyclonal anti-FD-MLO rabbit antibodies, then with gold-conjugated anti-rabbit IgG. Labeling was revealed using the silver enhancement technique for light microscopy. MLO in frozen thin sections of glands were efficiently labeled. Optimal results were obtained with 4% paraformaldehyde-0.1% glutaraldehyde fixation and low-temperature embedding in LR White resin. Both scattered MLO and unusual dense forms of MLO were easily detected with the electron-dense gold probe. This method distinguished MLO from other membrane-limited bodies and provided a good tool for studying infection in large regions of FD-infected tissues by light microscopy.     

Dependence of the elastic characteristics and contractile activity of the rat tail artery on the transmural difference of potentials

Change in the diffuse portion of the double electric layer at the blood-vascular wall border under the effect of thorium nitric oxide and heparin affects the tone and contractile ability of the rat tail artery: thorium diminishes the vessel rigidity and the amplitude of its contractile response, whereas heparin augments the rigidity as well as the amplitude of the response. Thorium nitric oxide also diminishes rigidity of the vessel connective tissue skeleton. The difference of potentials between the vessel lumen and its wall seems to be capable of affecting the contractile apparatus of the smooth muscle cells by means of activation of potential-sensitive ionic channels.     

A new method for transfer of polyethylene glycol-embedded tissue sections to silanated slides for immunocytochemistry

Polyethylene glycol (PEG) is an excellent embedding medium for immunohistochemical studies. It provides structural preservation superior to frozen sections and increased sensitivity of antigen detection compared with paraffin sections. One limitation of PEG embedment is that PEG sections are difficult to handle and adhere poorly to glass slides. Here we present a simple and effective method for embedding tissues in PEG and transferring the resultant sections onto silanated glass slides. In addition, a method for silver enhanced colloidal gold immunostaining was combined with common dye staining to demonstrate the excellent structure preservation and sensitive antigen detection. Bovine chorionic membrane was fixed with Bouin's fixative, embedded in polyethylene glycol (PEG) 1500, cut into 5-microns sections, flattened over agarose blocks (10 x 10 x 2 mm3), and blotted onto Digene silanated slides. Slides were then washed in PBS, which removed the PEG and agarose blocks. Tissue sections were immunocytochemically stained with dilute antiserum raised in a rabbit against purified bovine placental retinol binding protein (bpRBP). Sections were washed and incubated with 1-nm colloidal gold-labeled goat anti-rabbit IgG. The immunogold particles were enhanced by silver staining (IGSS). Specimens were observed and photographed with an Olympus epipolarization microscope. The new method offered excellent morphological preservation of cell structure and the epipolarization microscopy provided high sensitivity for detection of specific immunogold-silver particles.     

A Modified Silver Impregnation Technique for the Observation of Thick Sections of Lymph Nodes Perfused with Colloidal Carbon

For many years, a variant of the silver impregnation technique of Bielchowsky has been used to study the lymph node because it clearly outlines the various structures which are usually hard to contrast with standard staining methods. Like other variants of silver impregnation, this method blackens the cell nuclei as well as the reticular fibers; however, it inhibits the impregnation of the nuclear chromatin immediately adjacent to fibers. Hence, this variant selectively darkens the lymphoid cell populations of the nodal structures which contain a loose fiber network.

To study the blood vascular network of the lymph node based on perfusion of colloidal carbon, a staining procedure was needed which would contrast nodal structures on thick sections, while allowing the carbon-filled small blood vessels to be distinguished from the impregnated coarse reticular fibers. In an attempt to adapt this variant of Bielchowsky's technique, 10, 20, 40 and 60 nm thick sections from rat nodes, fixed in a solution of Bouin-Hollande for 72 hr, were silver impregnated with serial dilutions (1:2 to 1:128) of the ammoniacal silver solution. Forty-micrometer thick sections impregnated with a 1:16 dilution of the original silver solution at 37 C and for 30 min provided the best results for the conditions.     

A combined staining method for argyrophilic nucleolar organizer regions and for glial fibrillary acidic protein in astrocytes of human brain

Summary Different protocols are described for the combined staining method by which argyrophilic nucleolar organizer region sites can be evaluated in human astrocytes that are immunoreactive for glial fibrillary acidic protein. Among the four protocols studied, the following method was superior to others in terms of unambiguous visualization of the regions in glial fibrillary acidic protein-positive astrocytes; the first step was immunostaining for the protein with a blue colour reaction of alkaline phosphatase, followed by sequential colloidal silver staining for the regions. By this double staining method, we have demonstrated that the reactive astrocytes found in white matter around the metastatic lesion of carcinoma and the infarction, contain more argyrophilic nucleolar organizer regions in terms of the count as well as the area than glial fibrillary acidic protein-positive astrocytes present in the white matter of the normal brain. In conclusion, the double staining may provide valuable information on the cellular activity of astroglia when performed on routine formalin-fixed paraffin sections of the human brain.     

Localization of immunoreactive tyrosine hydroxylase in the goldfish retina with pre-embedding immunolabeling with one-nanometer colloidal gold particles and gold toning.

The goal of this study was to develop an alternative to silver intensification for visualizing small colloidal gold particles by light and electron microscopy. The isolated goldfish retina was labeled with rabbit antiserum to tyrosine hydroxylase and 1-nm colloidal gold-conjugated goat anti-rabbit IgG. The gold particles were enlarged by toning with gold chloride, followed by reduction in oxalic acid. Dopaminergic interplexiform cells were clearly visible by light microscopy and, in lightly-fixed material treated with detergent, they were labeled in their entirety. Labeling was qualitatively similar, although less extensive, in material fixed and processed for electron microscopy. The labeled processes were apparent in ultra-thin sections viewed at low magnification, but the gold-toned particles were not so large that they obscured subcellular structures. The procedure apparently had no deleterious effects on the tissue, since the ultrastructural preservation was comparable to that seen with other pre-embedding immunolabeling methods. The technique was simple, reliable and, since the gold solutions were so dilute, relatively inexpensive.     

Applications of immunocolloids in light microscopy. IV. Use of photochemical silver staining in a simple and efficient double-staining technique

We report the development of a new light-microscopic double-staining technique using colloidal gold as sole marker. The contrasting color to the red of colloidal gold is achieved by the application of photochemical silver reaction. The silver reaction, which is principally performed at the end of the first staining sequence, converts the red color of a gold-labeled reagent into black. This contrasts clearly with the red coloration that results from the second incubation sequence without silver reaction. For antigen double staining, the same protein A-gold complex can be used to provide the black and the red color, thus rendering the technique very economical. Alternatively, combination of protein A-gold immunolocalization and lectin-gold staining is possible, as is combined lectin-gold staining.     

Segmental differentiations of cell junctions in the vascular endothelium. The microvasculature

Small vascular units consisting of an arteriole, its capillaries, and the emerging venule (ACV units) were identified in the rat omentum and mesentery. They were fixed in situ and processed for electron microscopy either as whole units or as dissected segments. Systematic examination of the latter (in thin sections, as well as in freeze-cleaved preparations) showed that the intercellular junctions of the vascular endothelium vary characteristically from one segment to another in the microvasculature. In arterioles, the endothelium has continuous and elaborate tight junctions with interpolated large gap junctions. The capillary endothelium is provided with tight junctions formed by either branching or staggered strands; gap junctions are absent at this level. The pericytic venules exhibit loosely organized endothelial junctions with discontinuous low-profile ridges and grooves, usually devoid of particles. No gap junctions were found in these vessels. The endothelium of muscular venules has the same type of junctions (discontinuous ridges and grooves of low profile); in addition, it displays isolated gap junctions of smaller size and lower frequency than in arterioles. The term communicating junction (macula communicans) is proposed as a substitute for gap junctions, since the latter is inappropriate, in general, and confusing in the special case of the vascular endothelium.