Rapid-freezing cytochemistry: preservation of tubular lysosomes and enzyme activity

We show that tubular structures present in phorbol ester-stimulated macrophages are sensitive to commonly used chemical fixatives (i.e., they usually become fragmented during fixation). These structures are well preserved in macrophages that are physically fixed by rapid-freezing and subsequent freeze-substitution in osmium-acetone. We have developed methods that combine rapid-freezing, freeze-substitution, and enzyme cytochemistry for preservation of these tubular structures and for detection of endocytosed material (i.e., horseradish peroxidase). This method of rapid-freeze cytochemistry may be useful in other situations where chemical fixation does not adequately preserve cell structures, particularly of membrane compartments.     

Dentin sialoprotein: biosynthesis and developmental appearance in rat tooth germs in comparison with amelogenins,osteocalcin and colagen type-I

A non-collagenous protein, extracted from rat incisor dentin, is a dentin sialoprotein (DSP). We examined immunohistochemically the developmental appearance and tissue distribution of DSP in 1 to 3-day-old rat molar and incisor tooth germs. The earliest staining for DSP was observed in newly differentiated odontoblasts. In more advanced stages, immunostaining for DSP gradually increased in pre-dentin, odontoblasts and dentin, and appeared in many cells of the dental papilla. In early stages of development before the breakdown of the dental basement membrane, pre-ameloblasts were also positive for DSP. This staining disappeared from the ameloblast cell body soon after deposition of the first layer of mineralized dentin. Radiolabelling of tooth matrix proteins with ¹⁴C-serine in vitro followed by immunoprecipitation and fluorography confirmed that DSP was synthesized by tooth-forming cells. The immunolocalization for DSP was different from that of either collagen type-I, osteocalcin or the amelogenins. Whereas collagen type-I and osteocalcin were restricted to the mesenchymal dental tissues, the amelogenins were detectable in both epithelial and mesenchymal dental cells and tissues at the epithelio-mesenchymal interface at early stages of development, prior to the onset of dentin mineralization. We conclude that DSP is expressed in and secreted by odontoblasts and some dental papilla cells from early stages of dentinogenesis onwards, i.e. later than type-I collagen, but before deposition of the first layer of mineralized dentin. In pre-mineralizing stages, some of the matrix proteins may be endocytosed from the pre-dentin by both cell types involved in the epithelio-mesenchymal interaction.     

Localization of phosphophoryn in rat incisor dentin using immunocytochemical techniques

We studied the distribution of the phosphophoryn present in rat incisors by immunolocalization and histochemical techniques. The polyclonal antibody used reacts with both phosphorylated and de-phosphorylated phosphophoryn. Technical problems encountered in immunostaining and in preparing sections from mineralized dentin were resolved by use of peroxidase-conjugated protein A as the "second antibody" in indirect immunostaining reactions and by surface etching of partially demineralized sections. Staining with anti-rat incisor alpha-phosphophoryn antibody showed light staining over the odontoblasts and proximal odontoblastic processes, no stain over the predentin, dense staining over the intertubular dentin, and no stain over the mantle dentin. In the intertubular dentin the stain intensity was directly related to the distribution of mineral. These findings were directly corroborated by staining with Stains All. The mineralization of dentin and the distribution of phosphophoryn within the dentin may be much less uniform than previously supposed.     

Developmental expression of a 53 KD dentin sialoprotein in rat tooth organs.

Rat dentin contains a major sialic acid-rich glycoprotein, DSP, with an overall composition similar to that of bone sialoproteins but whose biological role in dentinogenesis is unknown. Using polyclonal affinity-purified antibodies to rat DSP and four immunohistochemical methods of detection, we studied the cell and tissue localization of DSP and the time course of its appearance during odontoblast differentiation. DSP first appeared within young odontoblasts concomitant with early secretion of pre-dentin matrix and before the onset of mineralization but was absent in pre-odontoblasts. DSP immunostaining also localized within secretory odontoblasts and was intense in odontoblastic processes. Early pre-dentin stained positive for DSP, in contrast to more mature pre-dentin, where immunoreactivity was less intense and more restricted to odontoblastic processes. In the zone of mineralized dentin matrix, a moderate and uniform staining pattern was evident. Intense immunostaining was also seen within the cells and matrix of dental pulp during dentinogenesis. Other cells and tissues within the tooth organ and those surrounding it were non-reactive. These findings suggest that DSP is developmentally expressed in cells of the odontoblastic lineage and may be a biochemical marker of odontoblastic activity.     

Immunohistochemistry of the intercellular matrix components and the epithelio-mesenchymal junction of the human tooth germ

Summary The immunohistochemical localization of heparan sulphate, collagen type I, III and IV, laminin, tenascin, plasma- and cellular fibronectin was studied in tooth germs from human fetuses. The lamina basalis ameloblastica or membrana preformativa, which separates the pre-ameloblasts from the pre-dentin and dentin, contained heparan sulphate, collagen type IV, laminin and fibronectin. Enamel reacted with antifibronectin, but the reaction varied depending on the type of fibronectin and the source of antibody. In early pre-dentin, collagen type I, laminin, tenascin and fibronectin were present. In late pre-dentin and dentin collagen type I was found in intertubular dentin and in the zone between enamel and dentin. The close relationship between collagen type I in dentin and fibronectin in immature enamel is interesting, as it may contribute to the stabilization of the amelodentinal interface. In dental pulp, collagen type IV and laminin were found in the endothelial basement membranes. Collagen type I and III, tenascin and fibronectin were localized to the mesenchymal intercellular matrix.The results of this study have supported the assumption that the lamina basalis ameloblastica is a basement membrane, and have lead to the suggestion that ameloblasts are producers of fibronectin or a fibronectin-like substance.     

Use of colloidal gold and ruthenium red in stereo high-voltage electron microscopic study of Con A-binding sites in mouse macrophages

Concanavalin A (Con A)-binding sites were labeled with colloidal gold (CG), stained with ruthenium red, and observed under a high-voltage electron microscope. Mouse peritoneal macrophages were labeled by the indirect Con A/CG labeling method at 0 degree C. After washing, some of the cells were incubated in phosphate-buffered saline (PBS) at 37 degrees C. The specimens were then stained with ruthenium red, to enhance the contrast of the cell surface, and embedded in Epon. Sections (0.3 approximately 3 micron thick) were cut and examined by high-voltage electron microscopy at accelerating voltages of 200 approximately 1,000 kV. Staining with ruthenium red provided a strong contrast of the cell surface and the invaginating tubules beneath it against the cytoplasm; in thick sections, both of them were clearly seen by stereomicroscopy. CG particles which represented Con A-binding sites were also sufficiently electron dense to be recognized by high-voltage electron microscopy of thick sections. The two- and three-dimensional distribution of CG particles on the ruthenium-red-positive cell surface was clearly visualized. At 0 degree C, Con A-binding sites were randomly distributed on the cell surface. The redistribution and endocytosis of Con A-binding sites were seen at 37 degrees C. The three-dimensional organization of membrane invagination, which represented the process of endocytosis, was clearly seen by stereomicroscopy. The combination of CG labeling and ruthenium red staining is a useful method for high-voltage electron microscopic analysis of the two- and three-dimensional distribution of CG-labeled ligands on the cell surface in thick sections.     

The ultrastructure of anionic sites in rat articular cartilage as revealed by different preparation methods and polyethyleneimine staining

The ultrastructure of anionic sites in the middle layer of rat articular cartilages was studied by two methods, the quick-freezing and deep-etching method, and the quick-freezing and freeze-substitution method. The anionic sites were visualized with a cationic tracer, polyethyleneimine. They were also compared with those revealed in tissues subjected to conventional fixation, such as pre-embedding or post-embedding. With the deep-etching method, three-dimensional meshwork structures were observed more clearly in the extracellular matrix compared with those seen in conventional ultrathin sections. In combination with polyethyleneimine staining, in which no chemical contrast was needed for visualization of anionic sites, numerous stained particles were detected around filaments in the extracellular matrix, indicating that they were anionic sites consisting mainly of proteoglycans. With the pre-embedding method and polyethyleneimine staining, the shapes of aggregated stained particles varied with different preparation procedures, including chemical fixation and contrasting. The fine meshworks were also observed with the post-embedding method and polyethyleneimine staining. It is suggested that such images of anionic sites, as revealed by the deep-etching method and the post-embedding polyethyleneimine-staining method with low-temperature dehydration, are probably closer to native states than those revealed by the conventional pre-embedding polyethyleneimine-staining method. © 1998 Chapman & Hall     

Electron microscopy of malachite green--glutaraldehyde fixed bacteria.

Malachite green combined with glutaraldehyde has been used recently as a fixative for preserving and revaling lipid complexes in thin sections of eukaryotic cells examined by electron microscopy. When bacteria were prefixed with the above mixture granular electron dense inclusions were revealed in all cultures tested. These inclusions were replaced by electron transparent areas in cells fixed with glutaraldehyde alone. The structures were frequently located near to or within the nucleoid and adjacent to the cell membrane in Gram-negative bacteria and were associated with the nucleoid and mesosomes in Gram-positive species. Polyhydroxybutyrate granules, generally poorly preserved in thin sections of Aquaspirillum serpens, were well preserved by the malachite green-glutaraldehyde fixative. Malachite green complexes were observed outside of the cells in all preparations. Capsules were neither preserved nor stained.     

Electron Microscopy of Malachite Green— Glutaraldehyde Fixed Bacteria

Malachite green combined with glutaraldehyde has been used recently as a fixative for preserving and revealing lipid complexes in thin sections of eukaryotic cells examined by electron microscopy. When bacteria were prefixed with the above mixture granular electron dense inclusions were revealed in all cultures tested. These inclusions were replaced by electron transparent areas in cells fixed with glutaraldehyde alone. The structures were frequently located near to or within the nucleoid and adjacent to the cell membrane in Gram-negative bacteria and were associated with the nucleoid and mesosomes in Gram-positive species. Polyhydroxybutyrate granules, generally poorly preserved in thin sections of Aquaspirillum serpens, were well preserved by the malachite green-glutaraldehyde fixative. Malachite green complexes were observed outside of the cells in all preparations. Capsules were neither preserved nor stained.     

Differential Stain for acid fast Bacteria and Spores

A modified method of staining acid-fast organisms is described. After staining with carbol-fuchsin as usual in the Ziehl-Neelson method, wash with water and while the slide is still wet cover with a saturated acetone solution of malachite green for three to five minutes. Wash and examine. The acid-fast organisms and spores are red in a green background. If the smear is thick and appears too dense, dry for three minutes and hold over the mouth of a bottle of ammonia until decolorized to suit. Upon exposure to the air the green returns. This can be prevented by keeping the smear alkaline, by the addition of sodium bicarbonate.

A second method is described for use with sputum in which acid-fast organisms are scarce. It permits the examination of thick smears and therefore increases the chances of finding tubercle organisms when few in number. Stain with carbol fuchsin as in the Ziehl-Neelson method. Decolorize with 30% phenol-disulfonic acid in water for a few seconds or until decolorized. Wash and examine at once. If color returns upon washing decolorize again. The tubercle organisms appear red in a colorless background.     

Colocalization of luciferin binding protein and luciferase to the scintillons ofGonyaulax polyedra revealed by double immunolabeling after fast-freeze fixation

Summary Two proteins,Gonyaulax luciferase and the luciferin binding protein, are involved in the bioluminescent reaction of the unicellular marine algaGonyaulax polyedra. Using antibodies raised separately against the purified proteins, their ultrastructural localizations were visualized by double immunogold labeling on sections after fast-freeze fixation, freeze-substitution and embedding in Epon or in LR White. Gold particles of two sizes attached to the secondary antibodies allowed the two primary antibodies to be distinguished. The two colocalized to cytoplasmic densifications (scintillons), which occurred in close association with the vacuolar membrane near the periphery of the cell. They also occurred in the cytoplasm of the Golgi area, either over densifications without associated membranes (prescintillons), or as very small colocalizations not associated with any evident cytoplasmic differentiation. No other site of colocalization was observed, thus unambiguously establishing the ultrastructural identity of the bioluminescent organelles.Abbreviations FFF fast-freeze fixation - FS freeze-substitution - IGS immunogold staining - LBP luciferin binding protein - PBS phosphate buffered saline - TBS tris-buffered saline Dedicated to the memory of Professor Beatrice Marcy Sweeney     

Microtrabecular structure of the axoplasmic matrix: visualization of cross-linking structures and their distribution

Axoplasmic transport is a dramatic example of cytoplasmic motility. Constituents of axoplasm migrate as far as 400 mm/d or at approximately 5 micron/s. Thin-section studies have identified the major morphological elements within the axoplasm as being microtubules, neurofilaments (100-A filaments), an interconnected and elongated varicose component of smooth endoplasmic reticulum (SER), more dilated and vesicular organelles resembling portions of SER, multivesicular bodies, mitochondria, and, finally, a matrix of ground substance in which the tubules, filaments, and vesicles are suspended. In the ordinary thin-section image, the ground substance is comprised of wispy fragments which, in not being noticeably tied together, do not give the impression of representing more than a condensation of what might be a homogeneous solution of proteins. With the high-voltage microscope on thick (0.5-micron) sections, we have noticed, however, that the so- called wispy fragments are part of a three-dimensional lattice. We contend that this lattice is not an artifact of aldehyde fixation, and our contention is supported by its visability after rapid-freezing and freeze-substitution. This lattice or microtrabecular matrix of axoplasm was found to consist of an organized system of cross-bridges between microtubules, neurofilaments, cisternae of the SER, and the plasma membrane. We propose that formation and deformation of this system are involved in rapid axonal transport. To facilitate electron microscope visualization of the trabecular connections between elements of axoplasm, the following three techniques were used: first, the addition of tannic acid to the primary fixative, OsO4 postfixation, then en bloc staining in uranyl acetate for conventional transmission electron microscope (TEM); second, embedding tissue in polyethylene glycol for thin sectioning, dissolving out the embedding medium from the sections and blocks, critical-point-drying (J. J. Wolosewick, 1980, J. Cell Biol., 86:675-681.), and then observing the matrix-free sections with TEM or the blocks with a scanning electron microscope; and third, rapid freezing of fixed tissue followed by freeze-etching and rotary- shadowing with replicas observed by TEM. All of these procedures yielded images of cross-linking elements between neurofilaments and organelles of the axoplasm. These improvements in visualization should enable us to examine the distribution of trabecular links on motile axonal organelles.     

Cytochemistry of kinetochores under electron microscopy

A cytochemical analysis has been performed on kinetochores of mouse, Allium and grasshopper under the electron microscope. The study was carried out using serial sections and cytochemical methods. Alcoholic PTA was used for basic protein staining and the EDTA method for preferential staining of ribonucleoproteins. In mouse and Allium chromosomes the kinetochore appears positively stained after PTA and EDTA. In grasshopper chromosomes, kinetochores appear as a fibrillar and less dense region and are positively stained after EDTA. Blocks from mouse treated with HCl prior to PTA stain show lower contrast in the kinetochore. When Allium cepa anthers were treated with RNase and perchloric acid (PCA) there was no positive effect after EDTA stain in the kinetochore region. It is suggested that non-DNA material takes part in the constitution of the kinetochore. This material would be made up, at least in part, of basic proteins and ribonucleoproteins.     

Reevaluation of envelope profiles and cytoplasmic ultrastructure of mycobacteria processed by conventional embedding and freeze-substitution protocols.

The cell envelope architectures and cytoplasmic structures of Mycobacterium aurum CIPT 1210005, M. fortuitum, M. phlei 425, and M. thermoresistible ATCC 19527 were compared by conventional embedding and freeze-substitution methods. To ascertain the integrity of cells during each stage of the processing regimens, [1-14C]acetate was incorporated into the mycolic acids of mycobacterial walls, and the extraction of labeled mycolic acids was monitored by liquid scintillation counting. Radiolabeled mycolic acids were extracted by both processing methods; however, freeze-substitution resulted in the extraction of markedly less radiolabel. During conventional processing of cells, most of the radiolabel was extracted during the dehydration stage, whereas postsubstitution washes in acetone yielded the greatest loss of radiolabel during freeze-substitution. Conventional embedding frequently produced cells with condensed fibrous nucleoids and occasional mesosomes. Their cell walls were relatively thick (approximately 25 nm) but lacked substance. Freeze-substituted cells appeared more robust, with well-dispersed nucleoids and ribosomes. The walls of all species were much thinner than those of their conventionally processed counterparts, but these stained well, which was an indication of more wall substance; the fabric of these walls, in particular the plasma membrane, appeared highly condensed and tightly apposed to the peptidoglycan. Some species possessed a thick, irregular outer layer that was readily visualized in the absence of exogenous stabilizing agents by freeze-substitution. Since freeze-substituted mycobacteria retained a greater percentage of mycolic acids in their walls, and probably other labile wall and cytoplasmic constituents, we believe that freeze-substitution provides a more accurate image of structural organization in mycobacteria than that achieved by conventional procedures.     

Ultrastructure of the synaptic ribbons in photoreceptor cells of Rana catesbeiana revealed by freeze-etching and freeze-substitution

Summary The three-dimensional structure of synaptic ribbons in photoreceptor cells of the frog retina was studied with freeze-etching and freeze-substitution methods, combined with a rapid-freezing technique. Although the synaptic ribbon consisted of two electron-dense plaques bisected by an electron-lucent layer in conventional thin sections, such lamellar nature was not so evident in freeze-etched replicas. The cytoplasmic surfaces of the synaptic ribbon presented an extremely regular arrangements of small particles 4–6 nm in diameter. Fine filaments 8–10 nm in diameter and 30–50 nm in length connected synaptic vesicles and the ribbon surface. These connections were mediated by large particles on both ends of the filaments. Approximately 3–5 filaments attached to one synaptic vesicle. Synaptic ribbons were anchored to a characteristic meshwork underlying the presynaptic membrane via another group of similar fine filaments. The meshwork seemed to be an etched replicated image of the presynaptic archiform density observed in thin sections.     

Cerium as amplifying agent--an improved cerium-perhydroxide-DAB-nickel (Ce/Ce-H2O2-DAB-Ni) method for the visualization of cerium phosphate in resin sections

A new visualization (Ce/Ce-H2O2-DAB-Ni) procedure for cerium (Ce III) phosphate in semithin and ultrathin plastic sections (Epon 812, Lowicryl K4M, glycol methacrylate) of rat kidney tissues that had been incubated before embedding for the demonstration of phosphatases (alkaline and acid phosphatase, 5(1)-nucleotidase, Mg-dependent ATPase) is described. For this purpose the hydrophobic Epon resin was removed in NaOH-ethanol solution, whereas the hydrophilic Lowicryl and methacrylate sections did not required any etching. The primary reaction product Ce III-phosphate was amplified in a Ce III-citrate solution, subsequently oxidized with H2O2 and then visualized in a H2O2 containing DAB-nickel medium (Ce IV-perhydroxy induced DAB polymerization principle). The method yielded a very clear localization of enzyme activity. The final reaction product (DAB-nickel polymers) in 0.5 - 2.0 microns semithin sections is blue-black; the background staining is completely prevented. An increase of the staining contrast was obtained by posttreatment with OsO4 (osmium black formation). Furthermore, the enzyme reaction product could be demonstrated in 40 nm thick ultrathin sections by silver intensification, which utilized the high argyrophilia of the polymerized DAB-nickel complexes. This procedure replaces the earlier published technique.     

Fine structures of the capsules of Klebsiella pneumoniae and Escherichia coli K1.

The fine structures of the capsules of Klebsiella pneumoniae and Escherichia coli were determined by the rapid-freezing technique. The capsular layer was seen as a densely packed accumulation of fine fibers. The thickness of the capsule was approximately 160 nm in K. pneumoniae and less than 10 nm in E. coli K1. Two layers were observed in the Klebsiella capsule in which the arrangements of the fibers were different. The inner layer of the capsule was formed by a palisade of thick and dense bundles of the fibers standing at right angles on the surface of the outer membrane. In the outer layer these thick bundles of fibers loosened into fine fibers which spread over the bacterial surface, forming a fine network structure.     

Dentine proteoglycans: composition, ultrastructure and functions

Summary Proteoglycans (PGs) have been visualized in the predentine and dentine with cationic dyes by staining thin sections with Alcian Blue, bismuth nitrate, or using Spicer's high-iron diamine (HID) method. The precise location may be obtained by adding cationic dyes such as Cuprolinic Blue, ruthenium hexammine trichloride or cationic detergent (cetylpyridinium chloride) to the fixative. These methods induced the formation of aggregates which varied in shape and number according to the method used. Rapid freezing followed by freeze-substitution revealed an amorphous ground substance, homogeneously stained with Alcian Blue, located in the predentine between the collagen fibres. These PGs may be involved in transport and diffusion in predentine. In dentine, small granules and needle-like structures were observed along the collagen fibres. This second group of PGs differs in composition, distribution and functions from the predentine PGs. The same distribution was seen when hyaluronidase-gold labelling was used. Labelling with antibodies and autoradiography also gave evidence of two distinct groups of PGs. In predentine, as an hydrated gel, PGs seems to act as mineral inhibitors, whereas immobilized on a surface, as seen at the dentine edge, they act as nucleating agents. The interaction between PGs and phospholipids seems also to play a role in the mineralization process.     

Use of colloidal gold and ruthenium red in stereo high-voltage electron microscopic study of Con A-binding sites in mouse macrophages

Summary Concanavalin A (Con A)-binding sites were labeled with colloidal gold (CG), stained with ruthenium red, and observed under a high-voltage electron microscope. Mouse peritoneal macrophages were labeled by the indirect Con A/CG labeling method at 0° C. After washing, some of the cells were incubated in phosphate-buffered saline (PBS) at 37° C. The specimens were then stained with ruthenium red, to enhance the contrast of the cell surface, and embedded in Epon. Sections (0.33 m thick) were cut and examined by high-voltage electron microscopy at accelerating voltages of 2001,000 kV. Staining with ruthenium red provided a strong contrast of the cell surface and the invaginating tubules beneath it against the cytoplasm; in thick sections, both of them were clearly seen by stereomicroscopy. CG particles which represented Con A-binding sites were also sufficiently electron dense to be recognized by high-voltage electron microscopy of thick sections. The two- and three-dimensional distribution of CG particles on the ruthenium-red-positive cell surface was clearly visualized. At 0° C, Con A-binding sites were randomly distributed on the cell surface. The redistribution and endocytosis of Con A-binding sites were seen at 37° C. The three-dimensional organization of membrane invagination, which represented the process of endocytosis, was clearly seen by stercomicroscopy. The combination of CG labeling and ruthenium red staining is a useful method for high-voltage electron microscopic analysis of the two- and three-dimensional distribution of CG-labeled ligands on the cell surface in thick sections.     

Regular, rodlike, macromolecular structures in Artemia salina.

Artemia salina of different origins and in different developmental stages contains regular, rodlike, macromolecular structures. The particles were visualized by electron microscopy after negative staining. They can be purified from the postmitochondrial supernatant by sucrose gradient centrifugation in the presence of EDTA. Their size distribution has been measured from electron micrographs. The particles are probably nucleoprotein complexes.