Colloidal Staining Research Articles

アフィニティークロマトグラフィーと白金コロイド染色法を組み合わせたタンパク質の特異町高感度定量法

アフィニティークロマトグラフィーと白金コロイド染色法を組み合わせたタンパク質の特異町高感度定量法

Nucleolar organizer regions in glioma

Nucleolar organizer regions (NOR) are loops of ribosomal DNA(rDNA) which are transcribed by RNA polymerase I. They produce ultimately ribosome and protein. Thus they are believed to reflect nuclear activity. We applied silver colloid staining technique to human glioma to examine relationship between the mean number of Ag-NOR and histopathological grading. The mean number of Ag-NOR( S.E of the mean)were in normal brain, in astrocytoma, in malignant astrocytoma, and in glioblastoma multiforme. And there was a statistically significant difference among these. The results show that Ag-NOR technique is a rather simple and rapid method and will become a helpful tool for estimation of the proliferative potential of glioma.

Nucleolar organizing regions (NORs) in pleomorphic adenomas of the salivary glands

The number of nucleolar organizing regions (NORs) can be used to reveal the degree of cell activity or metabolism in histopathology specimens. Forty-one cases of pleomorphic adenomas of the salivary glands were studied with a silver nitrate colloid staining method to demonstrate the NORs in the cells of the solid and duct pattern areas and in chondroid areas. Acinic and intercalated ducts cells of the surrounding normal salivary glands were also examined. The data was analyzed statistically and it was found that the cells in the solid/ductal areas had a higher cellular activity than the chondroid cells. This difference is probably directly related with the possibility of malignant transformation and the incidence of the different types of malignant pleomorphic adenomas. Furthermore, it is concluded that the number of NORs in pleomorphic adenomas to define malignancy should be done discriminating between the different histologic patterns because of their different metabolic activities.

Silver colloid staining technique for analysis of glioma malignancy

A silver colloid staining technique for identifying nucleolar organizer region-associated proteins (Ag-NOR's) was applied to 51 human gliomas. These comprised 20 glioblastomas multiforme, 15 anaplastic astrocytomas, and 16 astrocytomas, in which the mean numbers of Ag-NOR's per cell (+/- standard error of the mean) were 2.51 +/- 0.12, 2.01 +/- 0.10, and 1.76 +/- 0.06, respectively. Significant differences among these were recognized, and the mean number of Ag-NOR's paralleled the degree of histopathological malignancy. In 16 cases, studies were performed of the number of Ag-NOR's and the S-phase fraction by in vitro labeling using antibromodeoxyuridine monoclonal antibody. A linear relationship was demonstrated between these two factors (r = 0.857, p less than 0.001), although some scatter was seen. In 32 adult patients, the correlation between the number of Ag-NOR's and the prognosis was estimated. The results demonstrated that the group containing patients with less than 1.80 Ag-NOR's per cell had a better prognosis than the group with 1.80 Ag-NOR's or more. Thus, the number of Ag-NOR's reflected the degree of histopathological malignancy, S-phase fraction, and prognosis. Silver colloid staining for Ag-NOR's is a simple, rapid, and reproducible method for estimating the proliferative potential of human gliomas without requiring a complicated technique.

Essential problems in quantification of proteins following colloidal staining with Coomassie Brilliant Blue dyes in polyacrylamide gels, and their solution

Quantitative determination of stained proteins following polyacrylamide gel electrophoresis (PAGE) is of increasing interest especially since computer-aided densitometers have become available as well as recipes for sensitive and background-free staining with Coomassie Brilliant Blue dyes. However, avoidance of separation artifacts is not the only essential prerequisite for quantitative evaluation. The local particle density of a protein in a given gel is of critical importance since it determines its stainability. Depending on local protein concentration, the dye binding to the same amount of a given protein differs considerably. Since the stainability of proteins using colloidal staining procedures, as with Coomassie Brilliant Blue dyes, is time-dependent and, in addition, also dependent on the pore size of a given polyacrylamide gel used for PAGE, calibration curves for quantitative determinations have to be prepared in polyacrylamide gels of the same composition as used for PAGE. Staining conditions also have to be identical for calibration gels and gels under analysis. If, however, a set of calibration curves is prepared for different staining times, it is possible to calculate a generalized calibration curve, allowing for quantitative evaluation with flexible staining time. Furthermore, and in consequence of the implications due to particle density, quantitative determination via densitometry is only possible by determining the protein amount of each single measuring point (pixel) via its absorbance on the basis of a calibration curve. Since the particle density is inherent in a calibration curve, the final summation of the protein amount per pixel will give values close to reality.

ChemInform Abstract: Highly Sensitive Protein Detection by Colloidal Platinum Staining Method.

ChemInform Abstract: Highly Sensitive Protein Detection by Colloidal Platinum Staining Method.

白金コロイドを用いるタンパク質の高感度検出法

白金コロイドを用いるタンパク質の高感度検出法

Growth cone localization of neural cell adhesion molecule on central nervous system neurons in vitro.

Ultrastructural analysis of colloidal gold immunocytochemical staining and immunofluorescence microscopy has been used to study the presence of neural cell adhesion molecule (NCAM) on the surface of neuronal growth cones. The studies were carried out with cultures of rat hypothalamic and ventral mesencephalic cells, using morphology and expression of tyrosine hydroxylase, neurofilaments, and glial fibrillary acidic protein as differential markers for neurons and glia. NCAM was found on all plasmalemmal surfaces of neurons including perikarya and neurites. The density of NCAM varied for different neurons growing in the same culture dish, and neurons had at least 25 times more colloidal gold particles on their plasmalemmal membranes than astroglia. Of particular interest in the present study was a strong labeling for NCAM on all parts of neuritic growth cones, including the lamellar and filopodial processes that extend from the tip of the axon. The density of NCAM was similar on different filopodia of the same growth cone. Therefore, in situations where homophilic (NCAM-NCAM) binding might contribute to axon pathfinding, a choice in direction is more likely to reflect differences in the NCAM content of the environment, rather than the distribution of NCAM within a growth cone. On the other hand, the variation in NCAM levels between single neurons in culture was significant and could provide a basis for selective responses of growing neurites.

Histochemical detection of anionic components in the cephalopod brain

Brains obtained from three species of mediterranean cephalopods (Loligo, Sepia, Octopus) were fixed in Bouin's solution. Paraffin sections were cut sequentially at the frontal plane and used for Alcian blue staining (critical electrolyte concentration method), colloidal iron hydroxide staining procedure, the periodic acid Schiff's reagent method, and the lead tetra-acetate-Schiff method. The stained sections were evaluated at 2 regions of different histological composition: the palliovisceral ganglion and the optic lobe. A high concentration of anionic components was found in synaptic regions of the neuropil whereas neuronal cell bodies showed a relatively weak staining of these constituents. There was a significant reaction of the perineuronal glia nets in the cellular rind of the palliovisceral ganglion. From the comparison of staining patterns obtained with the 4 methods in this study and literature data it can be concluded that the detected anionic sites are mainly carboxyl groups of acidic proteins and/or glycoproteins. Sulphate groups may be present in lower concentrations. Their distribution reveals that the role of anionic components other than sialic acids in the invertebrate brain might be discussed in the context of synaptic transmission similar to that in vertebrates. The possible involvement of the glia cell population has to be taken into consideration.

Colloidal iron hydroxide staining of surface carbohydrates after glycerol treatment of uterine epithelial cells.

The structural relationships between intramembraneous particles (IMPs) and surface carbohydrates has been studied in uterine epithelial cells with a colloidal iron hydroxide (CIH) technique. To aggregate IMPs, glycerol treatment of unfixed cells was used and this treatment also caused some patching of CIH deposits on the cell surface. We conclude that some of the CIH receptors may be the surface expression of the IMPs.

Wheat germ agglutinin indicates the commencement of neuronal differentiation in the embryonic brain. A comparative carbohydrate histochemical study

Paraffin sections prepared from prenatal rat brain (embryonic days 14, 16, 18 and 20) were incubated with buffered wheat germ agglutinin labelled with fluorescein isothiocyanate (FITC-WGA). In the embryonic brain, the proliferating cells of the ventricular zone showed a low binding of WGA. Differentiating neuroblasts and axonal bundles exhibited a significantly higher degree of labelling. This pattern was most evident in the cerebral cortex where the marginal zone and the sub-plate region were found to be most intensely strained. The structural basis of WGA-binding was specified by comparison with data obtained by the colloidal iron hydroxide staining procedure, the periodic acid Schiff's reagent method and Alcian Blue staining (critical electrolyte concentration method). The results show that at regions of intensive differentiation in the embryonic brain there is an increase in the concentrations of sialic acid and glycosaminoglycans.

Clonidine after renal ischemia to lessen acute renal failure and microvascular damage

Clonidine, an antihypertensive drug that inhibits renin release and causes a water diuresis in normal animals, was tested for its ability to reduce the severity of post-ischemic acute renal failure produced in rabbits by clamping the left renal pedicle for 1 hour and removing the opposite kidney. Clonidine significantly lessened renal failure when given during, or 1 hours after, the ischemic insult in dehydrated rabbits. It was also effective when given during the ischemic insult in vasopressin-treated water-drinking rabbits but not in control water-drinking rabbits. In vasopressin-treated rabbits, clonidine lessened renal failure observed 2 days after the ischemic insult despite the fact that in the immediate postischemic period it lowered total renal blood flow, produced hypotension, and did not bring about lower plasma renin levels. Clonidine treatment resulted in less outer medullary microvascular damage (demonstrated by colloidal carbon staining), higher outer medullary blood flow 1 to 2 hours after unclamping, fewer casts, and higher creatinine clearance and free water clearance/creatinine clearance 4 to 6 hours after unclamping compared with controls. The effect of clonidine was unrelated to plasma renin activity. Clonidine did not alter plasma vasopressin concentration. Demeclocycline and lithium, two agents that blunt renal responsiveness to vasopressin, had a beneficial effect in dehydrated animals similar to that of clonidine, but the angiotensin II antagonist saralasin and the angiotensin converting enzyme inhibitor SQ20881 did not. Normal rabbits given a large dose of vasopressin in oil plus clonidine had significantly greater urine output and free water clearance and lower urine osmolality than did rabbits given vasopressin in oil alone. These results suggest that clonidine may be beneficial because it prevents ischemic microvascular injury in the renal outer medulla, an effect that may decrease tubular obstruction by lessening desquamation of damaged tubular cells or cell constituents into the tubular lumen. Clonidine may also decrease formation of obstructive hyaline casts in collecting ducts by blunting the kidney's response to vasopressin and increasing tubular fluid flow rate.

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.

Surface properties of extracellular malaria parasites: morphological and cytochemical study.

Morphological and cytochemical surface characteristics of isolated malaria parasites (Plasmodium berghei) and host erythrocytes were compared by electron microscopy by using thin section and carbon replica techniques. Erythrocytes were uniform in shape and had fine, granular surfaces. In contrast, free parasites exhibited a variety of sizes, shapes, and surface textures. Fine surface stippling was a common topographical feature of isolated parasites. Small, infective forms often had patterned surfaces resulting from the protuberance of an underlying thick intermediate layer. Results of cytochemical analysis using a sialophilic colloidal iron stain indicated that the malaria parasite's surface lacked exposed sialic acid groups which would normally give rise to a net negative surface charge common to erythrocytes. Biochemical assay demonstrated that malaria parasites contained about one-half the amount of sialic acid per unit weight as did control red cell extracts. Similarly, external acidic mucopolysaccharide coats of free parasites, as revealed by ruthenium red staining were extremely thin as compared with the thick glycocalyx layer of red cells. Lipid plaques at the surface of parasites and red cells were localized by lipophilic iron colloid staining. Although the gross patchwork distribution of plaques was somewhat similar for the two cell types, the parasites were stained more intensely and had a closer-knit patchwork pattern than those exhibited by the erythrocytes. Such findings indicate that there are slight differences in the arrangement of phospholipids at the surfaces of limiting membranes of host cells and parasites. The significance of the above cytochemical surface properties of the malaria parasite (which are seemingly akin to those of intracellular organelles is discussed in relation to certain host-parasite interactions, such as parasite adhesion to target cells and enhanced clearance of extracellular parasites.

Assembly of lipid-containing viruses.

AbstractEnveloped viruses which form by budding at the cell surface possess a membrane consisting of a lipid bilayer and a small number of virus‐coded polypeptides. Since viral polypeptides become integral components of the plasma membrane during assembly, the process of synthesis and incorporation into membranes of these proteins may reflect the pathway of plasma membrane assembly. Electron microscopic studies have suggested that viral envelope proteins are incorporated into discrete, localized regions of the plasma membrane which serve as recognition sites for the viral nucleocapsid. In influenza virus‐infected cells, viral polypeptides are associated with cytoplasmic membranes as well as the plasma membrane. The major envelope glycoprotein appears to be synthesized in rough endoplasmic reticulum, and to migrate to smooth membranes after synthesis. Glycosylation is initiated in rough membranes and progresses further in smooth membranes. Unlike the glycoproteins, the major nonglycosylated polypeptide appears to be inserted directly into the plasma membrane. In the presence of 2‐deoxyglucose or high concentrations of glucosamine, aberrant viral glycoproteins are detected which appear to be unglycosylated or partially glycosylated; these are associated with membranes and incorporated into virus particles of reduced infectivity. Therefore normal glycosylation is not essential for incorporation of viral glycoproteins into cellular membranes or virus particles, but is required for normal biological activity. The role of the viral neuraminidase in assembly and release has been studied using mutants defective in neuraminidase at restrictive temperature. Under these conditions virus formation occurs, but the progeny form large aggregates at the cell surface. Colloidal iron hydroxide staining indicates that such virus particles contain neuraminic acid, and these residues appear to serve as receptors leading to the extensive aggregation.

Estrogen-induced surface coat and enzyme changes in the implanting mouse blastocyst

ABSTRACT Blastocysts from estrogen-stimulated mice tend to be very sticky, a characteristic which can be correlated with estrogen-induced enzyme activation and changes in the surface coat of the trophoblast cells. The blastocyst surface coat was investigated using colloidal iron-Prussian blue stain. In addition, the activity of tyrosine aminotransferase was assayed histochemically in preimplantation blastocysts. The surface and enzyme changes were correlated with the presence of estrogen by investigations in normal blastocysts from intact mice before and after the endogenous estrogen surge and in delayed-implantation blastocysts from ovariectomized mice preceding and following estrogen treatment. Colloidal iron stains the surface coat of the blastocyst after, but not before, the endogenous estrogen surge and the exogenous estrogen treatment. Tyrosine aminotransferase activity also is increased considerably after estrogen induction.

Fine structural study of osteocytes and their surrounding bone matrix with respect to their age in young chicks

These studies show that the changes in the fine structure of the osteocytes with their age are correlated with the alterations in the periosteocytic bone matrix. These changes seem to be functionally significant. A young, bone-forming cell gradually changes into a mature bone-resorbing cell. Finally it degenerates. The osteocyte population is thus heterogeneous and is constituted by functionally different cells. The peripheral region of the newly formed pericellular bone matrix is recognizable as perilacunar matrix and becomes mineralized. The matrix near the cell, remains as osteoid and is the pericellular space. Later these regions are occupied by either a completely broken down or a partially broken down (modified matrix) bone matrix, both devoid of mineral. The breakdown product as shown by colloidal iron and colloidal thorium staining techniques seems to be acid mucopolysaccharide in nature.

Cytological Studies on the Absorptive Surfaces of Cestodes. IV. Localization and Cytochemical Properties of Membrane-Fixed Cation Binding Sites

The tegument plasmalemma of Hymenolepis diminuta and Lacistorhynchus tenuis is coated with a layer of acidic anions capable of adsorbing cations at neutral pH both in vivo and in vitro. This layer is structurally manifest in H. diminuta as a thickening of the outer dense leaflet of the and in L. tenuis as a distinct hirsute investment immediately superficial to the trilaminate component of the plasmalemma. This structure is demonstrable cytochemically in both species by the reaction of the surface with acidic suspensions of colloidal iron, and with osmium following periodate oxidation and adsorption of thiocarbohydrazide. The presence of sialic acid is indicated by biochemical analysis and the observed effects of polylysine, diazomethane, and neuraminidase on colloidal metal staining of the plasmalemma surface. Additional species of acidic anions may also be located at the surface of the tegument, particularly at the distal extremities of the microtriches. An increasing body of biochemical and cytochemical evidence, reviewed by Revel and Ito (1967), Benedetti and Emmelot (1967), and Ito (1969), indicates that carbohydrates, as constituents of glycoproteins, mucopolysaccharides, or gangliosides, form integral structural components of cell surfaces. The physiological significance of this (Bennett, 1963) has, in most cases, not yet been clearly elucidated. However, by virtue of their ionogenic substitutions, these carbohydrates are polyelectrolytes and, as such, might be expected to influence the ionic composition of the immediate extracellular environment. Accordingly, the glycocalyx has been implicated in a number of plasma functions, including adsorptive, transReceived for publication 12 February 1970. * Supported by grants from the NH (AI 08673, 5 TI GM 669), NSF (GB 7276, GB 17992), and Career Development Award K04-AI 23449 (to RDL) from the U. S. Public Health Service (NIAID). port, enzymic, and immunological activities (e.g., Brandt, 1962; Wolfe, 1964; Glick and Githens, 1965; Emmelot and Bos, 1966; Apffel and Peters, 1970). Concepts formulated by Read and co-workers (Read, Rothman, and Simmons, 1963; Read, 1966) related to the mechanisms of molecular interchange between endoparasitic helminths and their hosts have stimulated much recent interest in the structure and chemistry of worm surfaces. Particular emphasis has been placed on the tegument of cestodes and acanthocephala. Lacking an alimentary tract, these forms utilize the body wall as the principal organ for transport of material to or from the environment. Electron microscope investigations of the tapeworm and acanthocephalan tegument have revealed complex structural specializations for amplifying free surface area (reviewed by Lee, 1965; Lumsden, 1966a; Wright and Lumsden, 1969). Mucosubstances constitute an integral part of the acanthocephalan body surface (Crompton and Lee, FIGURES 1-3. Surface ultrastructure of the cestode tegument. 1. Survey image of the peripheral body wall of Lacistorhynchus tenuis, illustrating the brush border (BB) of microvilli (microtriches) lining the free surface. The underlying matrix is filled with vesicles, granules, and mitochondria. Section stained with uranyl acetate and lead citrate. X 11,200. 2. Surface plasmalemma of Hymenolepis diminuta at the base of a microvillus (Mv). The trilaminate unit membrane is structurally asymmetric, the outer dense leaflet (o) appearing significantly thicker than the inner dense leaflet (i). Section stained with uranyl acetate and lead citrate. X 576,000. 3. Surface plasmalemma of L. tenuis delimiting a microvillus. The limits of the trilaminate component are indicated by the arrows (um); note the filamentous coat superficial thereto. The limits of this coat are indicated by the bracket at f. Section stained with uranyl acetate and lead citrate. X 216,000.

Thyroid function in male killifish, Fundulus heteroclitus, adapted to high and low temperatures and to fresh water and sea water

1. 1. Adult male killifish, F. heteroclitus, were acclimated for 5 weeks to fresh water and sea water at 20 and 28°C. Estimates of thyroid activity were made from thyroid cell height, colloid staining properties, the rate of loss of thyroidal I 131 and serum concentrations of BEI 131. 2. 2. Results indicate that the levels of activity follow the sequence: FW20⩽FW28<SW20<SW28. 3. 3. Some of the possible interactions of salinity and temperature are discussed.

Electrophoretic Patterns of Soluble Proteins in Tobacco Leaf During Curing

AbstractPolyacrylamide gel disc electrophoresis revealed at least 13 anodic protein bands in leaf extracts of seven tobacco (Nicotiana tabacum L.) strains representing Burley, Turkish, flue- and fire-cured types. Four major bands B, E, G, and J were common in all strains. Weak bands varied slightly in mobility and in number among tobacco types. Studies of cell fractions of Burley 21 leaves yielded information that bands A, C, D, G, and J are present in the cytoplasm, whereas all but bands B, E, L, and M appeared to be identical in the chloroplast, mitochondrial, and nuclear extracts. The resemblance in protein banding pattern among the organelles indicates similarity in their membrane proteins. Bands B, E, L, and M are associated with plasma membrane or cell wall. During simulated air-curing, a majority of bands diminished by the tenth day, whereas bands B, E, and G remained highly intense and the concentration of band C was reduced in cured leaves. Bands B, E, and G reacted positively with the periodic-acid Schiff's reagent (PAS) but negatively with the colloidal ion staining. Trypsin digestion abolished protein bands stained with amido black or the PAS reagent. Results suggest that bands B, E, and G are neutral or weakly acidic glycoproteins