Acrolein Vapor Fixation in Electron Microscopy of the Horny Layer

2015 ◽  
pp. 207-214
Author(s):  
M. Kawai ◽  
N. Yoshizawa ◽  
G. Imokawa ◽  
K. Okamoto ◽  
K. Toda
Keyword(s):  
Electron Microscopy ◽  
Horny Layer

Electron and Immunoelectron Microscopical Studies on the Extracellular Protease (Keratinase) of Dermatophytes

1982 ◽  
Vol 40 ◽  
pp. 158-159 ◽  
Author(s):  
Y. Sei ◽  
I. Takiuchi ◽  
M. Masutani
Keyword(s):  
Electron Microscopy ◽  
Human Skin ◽  
Human Hair ◽  
Phosphate Buffer ◽  
Proteolytic Enzyme ◽  
Microsporum Canis ◽  
Horny Layer ◽  
Double Immunodiffusion ◽  
Igg Antibody ◽  
Rabbit Igg

Dermatophytes, known to contain the proteolytic enzyme keratinase can grow in keratinized structures. Here we describe the digestion of the horny layer and the first localization of the enzyme by immunoelectronmicroscopy. Microsporum canis isolated from an outpatient was cultured in a medium of: human hair, 2.9g; glucose, 0.5g; MgSO4-7H2O, 0.06 g; thiamine 0.01g; pyridoxine 0.01 g; and inositol 0.05 g in one liter of 0.028 M phosphate buffer (pH 7.8). The enzyme was isolated from the medium and purified by chromatography. Human skin was incubated with the purified protease (2mg/ml) for 12 hrs at 37° C, and then processed for electron microscopy. Controls were incubated without enzyme. Using rabbit IgG antibody against the purified enzyme the IgG-Fab'-peroxidase complex was synthesized according to the method of Nakane and Kawaoi. That complex gave a single band in double immunodiffusion against purified protease and in immunoelectrophoresis at pH 4.0 and 9.3. Human hairs with fungi were removed from the keratin medium on the 12th day, fixed with periodate-lysin-paraformaldehyde for 24 hrs at 4°C. They were again washed in PBS, preincubated in 3,3'-diaminobenzidine (DAB) for 30 min at room temp., put in a Graham and Karnovsky medium containing DAB and H2 O2 as a substrate for 5 min at room temp., postfixed in 1% OSO4 in PBS for 1 hr at 4°C., and embedded in Epon.

Changes to desmosomal antigens and lectin-binding sites during differentiation in normal human epidermis: a quantitative ultrastructural study

1989 ◽  
Vol 92 (4) ◽  
pp. 667-677
Author(s):  
C.J. Skerrow ◽  
D.G. Clelland ◽  
D. Skerrow
Keyword(s):  
Electron Microscopy ◽  
Binding Sites ◽  
Light Microscope ◽  
Lectin Binding ◽  
Human Epidermis ◽  
Immunogold Electron Microscopy ◽  
Horny Layer ◽  
Subsequent Decrease ◽  
Normal Human ◽  
Lectin Binding Sites

During epidermal differentiation, desmosomes undergo a series of changes in their abundance, structure and properties, which has previously been defined by conventional electron microscopy and the use of antibodies to desmosomal proteins at the light-microscope level. Such changes in a major adhesive organelle would be expected to have a significant role in the maintenance of epidermal organization, and therefore require more detailed characterization. In the present study, modifications to certain desmosomal components in normal human epidermis have been located and quantified by immunogold electron microscopy. Antibodies to desmosomal protein dp3 and glycoprotein dg1 were used to label the cytoplasmic regions of the junctions and lectins concanavalin A (ConA) and wheat germ agglutinin (WGA) to probe the extracellular glycosylated material. Binding was measured at histologically defined levels and expressed as gold particles per microns of desmosome length (linear particle density: LPD). In addition, desmosome frequency, expressed as the percentage of the cell membrane length occupied by desmosomes, was measured. Highly significant changes in desmosome frequency, diameter and LPD were observed between epidermal strata and, in basal and upper horny cells, between different regions of the same cell surface. These parameters rose to a maximum in the spinous or granular layers: their subsequent decrease continued without interruption across the interface between the living and terminally differentiated horny layers. Remaining reactivity with antibodies, but not lectins, was almost completely abolished immediately before the final disintegration of the desmosome structure in the lower horny layer. In contrast, numerous large, highly immunoreactive desmosomes were retained up to the outer surface in the grossly thickened horny layer found in callus. Though the overall pattern of a rise followed by a fall was similar for all parameters measured, differences were observed between probes. Thus, the extent of the rise in available antigen between the lateral and apical surfaces of the basal cell was greater for dg1 than for dp3; the subsequent decrease in dp3 antigens in upper epidermal layers was more rapid than that for dg1, and changes to both antigens preceded those to lectin-binding sites. These results show that differences in desmosome frequency and in the size and antibody-binding characteristics of individual junctions underlie the heterogeneous distribution of desmosomal components within epidermis that is found by light-microscope immunocytochemistry. They further suggest that the disintegration of desmosomes within normal horny layer, which is an essential preliminary to desquamation, is the culmination of a sequence of events that begins in the upper living tissue and initially involves cytoplasmic components.(ABSTRACT TRUNCATED AT 400 WORDS)

Platinum Compounds as Stains for Electron Microscopy

1974 ◽  
Vol 32 ◽  
pp. 230-231
Author(s):  
S. K. Aggarwal ◽  
P. McAllister ◽  
R. W. Wagner ◽  
B. Rosenberg
Keyword(s):  
Electron Microscopy ◽  
Hela Cells ◽  
Uranyl Acetate ◽  
Sarcoma 180 ◽  
Platinum Compounds ◽  
Kb Cells ◽  
En Bloc ◽  
Human Lymphoma ◽  
Ultrathin Sections ◽  
Blue Coloration

Uranyl acetate has been used as an electron stain for en bloc staining as well as for staining ultrathin sections in conjunction with various lead stains (Fig. 1). Present studies reveal that various platinum compounds also show promise as electron stains. Certain platinum compounds have been shown to be effective anti-tumor agents. Of particular interest are the compounds with either uracil or thymine as one of the ligands (cis-Pt(II)-uracil; cis-Pt(II)-thymine). These compounds are amorphous, highly soluble in water and often exhibit an intense blue coloration. These compounds show enough electron density to be used as stains for electron microscopy. Most of the studies are based on various cell lines (human AV, cells, human lymphoma cells, KB cells, Sarcoma-180 ascites cells, chick fibroblasts and HeLa cells) while studies on tissue blocks are in progress.

Immune Electron Microscopy (IEM) of a Canine Adeno-Associated Virus

1974 ◽  
Vol 32 ◽  
pp. 256-257
Author(s):  
Gunter F. Thomas ◽  
M. David Hoggan
Keyword(s):  
Electron Microscopy ◽  
Cell Cultures ◽  
Complement Fixation ◽  
Hepatitis Virus ◽  
Wide Distribution ◽  
Immune Electron Microscopy ◽  
Adeno Associated Virus ◽  
Virus Like Particle ◽  
Dog Kidney ◽  
Green Monkeys

In 1968, Sugimura and Yanagawa described a small 25 nm virus like particle in association with the Matsuda strain of infectious canine hepatitis virus (ICHV). Domoto and Yanagawa showed that this particle was dependent on ICHV for its replication in primary dog kidney cell cultures (PDK) and was resistant to heating at 70°C for 10 min, and concluded that it was a canine adeno-associated virus (CAAV). Later studies by Onuma and Yanagawa compared CAAV with the known human serotypes (AAV 1, 2, 3) and AAV-4, known to be associated with African Green Monkeys. Using the complement fixation (CF) test, they found that CAAV was serologically related to AAV-3 and had wide distribution in the dog population of Japan.

High Voltage Electron Microscopy of Ion-Milled Dental Enamel

1974 ◽  
Vol 32 ◽  
pp. 60-61
Author(s):  
L. D. Ackerman ◽  
S. H. Y. Wei
Keyword(s):  
Electron Microscopy ◽  
High Voltage ◽  
Third Molar ◽  
Polarized Light ◽  
Dental Enamel ◽  
Longitudinal Section ◽  
Ion Milling ◽  
High Voltage Electron Microscopy ◽  
Voltage Electron ◽  
High Voltage Electron

Mature human dental enamel has presented investigators with several difficulties in ultramicrotomy of specimens for electron microscopy due to its high degree of mineralization. This study explores the possibility of combining ion-milling and high voltage electron microscopy as a means of circumventing the problems of ultramicrotomy.A longitudinal section of an extracted human third molar was ground to a thickness of about 30 um and polarized light micrographs were taken. The specimen was attached to a single hole grid and thinned by argon-ion bombardment at 15° incidence while rotating at 15 rpm. The beam current in each of two guns was 50 μA with an accelerating voltage of 4 kV. A 20 nm carbon coating was evaporated onto the specimen to prevent an electron charge from building up during electron microscopy.

Pathogenesis of Human Hair Defects

1974 ◽  
Vol 32 ◽  
pp. 12-13
Author(s):  
P.S. Porter ◽  
T. Aoyagi ◽  
R. Matta
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Human Hair ◽  
Standard Techniques ◽  
Scanning Electron

Using standard techniques of scanning electron microscopy (SEM), over 1000 human hair defects have been studied. In several of the defects, the pathogenesis of the abnormality has been clarified using these techniques. It is the purpose of this paper to present several distinct morphologic abnormalities of hair and to discuss their pathogenesis as elucidated through techniques of scanning electron microscopy.

Evaluation of the dark field method for large association of spread DNA

1978 ◽  
Vol 36 (2) ◽  
pp. 190-191
Author(s):  
Douglas C. Barker
Keyword(s):  
Electron Microscopy ◽  
Molecular Weight ◽  
Mitochondrial Dna ◽  
Extraction Method ◽  
Field Method ◽  
Dark Field ◽  
Kinetoplast Dna ◽  
Field Electron ◽  
Field Electron Microscopy ◽  
Dark Field Electron

A number of satisfactory methods are available for the electron microscopy of nicleic acids. These methods concentrated on fragments of nuclear, viral and mitochondrial DNA less than 50 megadaltons, on denaturation and heteroduplex mapping (Davies et al 1971) or on the interaction between proteins and DNA (Brack and Delain 1975). Less attention has been paid to the experimental criteria necessary for spreading and visualisation by dark field electron microscopy of large intact issociations of DNA. This communication will report on those criteria in relation to the ultrastructure of the (approx. 1 x 10-14g) DNA component of the kinetoplast from Trypanosomes. An extraction method has been developed to eliminate native endonucleases and nuclear contamination and to isolate the kinetoplast DNA (KDNA) as a compact network of high molecular weight. In collaboration with Dr. Ch. Brack (Basel [nstitute of Immunology), we studied the conditions necessary to prepare this KDNA Tor dark field electron microscopy using the microdrop spreading technique.

Quaternary structure of Limulus polyphemus hemocyanin

1978 ◽  
Vol 36 (2) ◽  
pp. 176-177
Author(s):  
T. Wichertjes ◽  
E.J. Kwak ◽  
E.F.J. Van Bruggen
Keyword(s):  
Electron Microscopy ◽  
Functional Properties ◽  
Horseshoe Crab ◽  
Temperature Jump ◽  
Quaternary Structure ◽  
Crystallographic Analysis ◽  
Limulus Polyphemus ◽  
Oxygen Binding ◽  
X Ray ◽  
Intact Molecule

Hemocyanin of the horseshoe crab (Limulus polyphemus) has been studied in nany ways. Recently the structure, dissociation and reassembly was studied using electron microscopy of negatively stained specimens as the method of investigation. Crystallization of the protein proved to be possible and X-ray crystallographic analysis was started. Also fluorescence properties of the hemocyanin after dialysis against Tris-glycine buffer + 0.01 M EDTA pH 8.9 (so called “stripped” hemocyanin) and its fractions II and V were studied, as well as functional properties of the fractions by NMR. Finally the temperature-jump method was used for assaying the oxygen binding of the dissociating molecule and of preparations of isolated subunits. Nevertheless very little is known about the structure of the intact molecule. Schutter et al. suggested that the molecule possibly consists of two halves, combined in a staggered way, the halves themselves consisting of four subunits arranged in a square.

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