A simple technique for supporting single cells in electron microscopic immunocytochemistry and embedding

Summary Cell suspensions of rat anterior pituitaries were filtered with a polycarbonate filter (pore size 3 m) and fixed on the filter. After fixation the cells were adherent to the filter and immunocytochemical staining could be accomplished by simply dipping the filter into the different incubation media. The cells could be dehydrated and embedded in Epon 812 on the filter. After polymerization the embedded filter was sawn into small blocks and the cell layer was sectioned tangentially on an ultramicrotome. This method also seems to be applicable to other histochemical studies on single cells.Supported by Deutsche Forschungsgemeinschaft, SFB 87/B2     

A Filter Method for Processing Small Samples of Marine Ova Through Fixation, Dehydration, and Embedding Without Changing Containers

Free-floating cells can be fixed, dehydrated, and embedded in a single container. The container was constructed from stainless steel, and the paraffin block formed by the shape and size of a container was perfect for microtoming. Eight containers were embedded in a fiberglass holder. This holder was designed so that it could be used with a 47 mm Millipore filter. Cells were pipetted into the top of a container while the Millipore filter sealed the bottom; thus the cells were retained on the filter while fluids were allowed to pass through it. The exposure of the cells to histological reagents was regulated by applying a vacuum to control the rate of flow through the filter.     

Direct Epoxy Embedding for Vertical Sectioning of Cells Grown as a Monolayer on Millipore Filter

Cells cultured as a monolayer on MF-Millpore GSWP. 0.22 μ pore size, filter were fixed, dehydrated, and examined by phase-contrast microscopy with the filter immersed in a 1:1 mixture of xylene and the embedding medium. The membrane was cut into 2 × 20 mm strips, and each strip which was selected for desired cells was embedded vertically in a BEEM capsule. Thus direct embedding which allowed edgewise sectioning of cells was obtained without removing them from the culturing support.     

A Rapid Technique for Preparing Microorganisms for Transmission Electron Microscopy

A rapid and efficient method of preparing microorganisms for transmission electors microscopy is reported. In developing the method Salmonella, streptococcal, ad protozoal specimens were fixed with glutaraldehyde. After fixation cells are collected on a membrane filter, washed with buffer, postfixed with osmium tetroxide, then washed with distilled water and stained en bloc with uranyl acetate. Specimens are dehydrated using a graded series of acetone and then infiltrated with graded mixtures of acetone and Spurr embedding medium. Finally the membrane filter is cut into small pieces and embedded in fresh embedding medium polymerized in polyethylene capsules. By collecting and processing the specimens on membrane filters, numerous centrifugations are eliminated from standard procedures. The use of a low viscosity embedding medium allows for rapid infiltration and embedding of the specimen. Using this technique microbial specimens can be sectioned after less than 4 hours preparation.     

A rapid technique for preparing microorganisms for transmission electron microscopy.

A rapid and efficient method of preparing microorganisms for transmission electron microscopy is reported. In developing the method Salmonella, streptococcal, and protozoal specimens were fixed with glutaraldehyde. After fixation cells are collected on a membrane filter, washed with buffer, postfixed with osmium tetroxide, then washed with distilled water and stained en bloc with uranyl acetate. Specimens are dehydrated using a graded series of acetone and then infiltrated with graded mixtures of acetone and Spurr embedding medium. Finally the membrane filter is cut into small pieces and embedded in fresh embedding medium polymerized in polyethylene capsules. By collecting and processing the specimens on membrane filters, numerous centrifugations are eliminated from standard procedures. The use of a low viscosity embedding medium allows for rapid infiltration and embedding of the specimen. Using this technique microbial specimens can be sectioned after less than 4 hours preparation.     

Nonvirus encoded proteins could be embedded into Bombyx mori cypovirus polyhedra

To explore whether the nonvirus encoded protein could be embedded into Bombyx mori cypovirus (BmCPV) polyhedra. The stable transformants of BmN cells expressing a polyhedrin (Polh) gene of BmCPV were constructed by transfection with a non-transposon derived vector containing a polh gene. The polyhedra were purified from the midguts of BmCPV-infected silkworms and the transformed BmN cells, respectively. The proteins embedded into polyhedra were determined by mass spectrometry analysis. Host derived proteins were detected in the purified polyhedra. Analysis of structure and hydrophilicity of embedded proteins indicated that the hydrophilic proteins, in structure, were similar to the left-handed structure of polyhedrin or the N-terminal domain of BmCPV structural protein VP3, which were easily embedded into the BmCPV polyhedra. The lysate of polyhedra purified from the infected transformation of BmN cells with modified B. mori baculovirus BmPAK6 could infect BmN cells, indicating that B. mori baculovirus could be embedded into BmCPV polyhedra. Both the purified polyhedra and its lysate could be coloured by X-gal, indicating that the β-galactosidase expressed by BmPAK6 could be incorporated into BmCPV polyhedra. These results suggested that some heterologous proteins and baculovirus could be embedded into polyhedra in an unknown manner.     

Interference of tooth differentiation with interposed filters

The effect of interposed Nuclepore filters on the epithelio-mesenchymal interaction in embryonic mouse tooth was studied. Filters with pore sizes of 0.6 and 0.2 μm allowed differentiation of odontoblasts and ameloblasts in the bell-stage tooth germ. This differentiation progressed more rapidly when the 0.6-μm pore size filter was used. Nuclepore filters with 0.1-μm pores prevented differentiation. Electron microscopic examination revealed penetration of cell processes into the filter pores. Cytoplasmic material could be seen in the 0.6-μm pore-size filter within 3 days of cultivation, whereas, in the 0.2-μm filter pores, penetration was slight. After 6 days of cultivation, cytoplasmic material was found at all levels of the 0.2-μm pore-size filter, but not in the channels of the 0.1-μm pore-size filters, preventing differentiation. It is concluded that the 0.1-μm pore-size filter blocks tooth development at the level of mesenchymal cell differentiation into odontoblasts. It is suggested that this differentiation requires a close association between the interacting mesenchymal and epithelial cells.     

Suction sampling technique for obtaining standardized cytologic specimens from the oral mucosa

To enhance the value of exfoliative cytology for the study of the oral mucosa, a simple apparatus was developed to permit adequate sampling of a specific site so that samples collected on different occasions could be compared. The device essentially consists of a collecting cup connected to a blood collection evacuation system. The collecting cup is a modified female half of a stainless-steel filter holder supporting a 13-mm-diameter cellulose filter of 0.05-microns pore size. Suction pressure is applied by means of a 10-mL glass tube of premeasured vacuum. After positioning the collecting cup on the selected site on the buccal mucosa, the vacuum (440 mm Hg) is applied for five seconds. The mucosa is drawn in against the filter, producing a monolayered imprint of cells. This sample may be disengaged from the filter by agitation into a solution; this allows quantitative cytologic studies, such as the measurement of cell numbers by an electronic counter or the estimation of the areas of cells and nuclei by computer-aided image analysis of Cytospin preparations. Five separate samplings from each of three test subjects produced a harvest of 3,000 to 7,000 epithelial cells per sample; the cellular areas ranged from 784 to 1,052 sq microns while the nuclear areas ranged from 18.4 to 21.8 sq microns.     

Model system for studies of microbial dynamics at exuding surfaces such as the rhizosphere

An autoclavable all-glass system for studying microbial dynamics at permeable surfaces is described. Standard hydrophobic or hydrophilic membranes (46-mm diameter) of various pore sizes were supported on a glass frit through which nutrient solutions were pumped by a peristaltic pump. The pump provided a precisely controlled flow at speeds of 0.5 to 500 ml of defined or natural cell exudates per h, which passed through the membrane into a receiving vessel. The construction allowed a choice of membranes, which could be modified. The system was tested with a bacterium, isolated from rape plant roots (Brassica napus L.), that was inoculated on a hydrophilic membrane filter and allowed to develop into a biofilm. A defined medium with a composition resembling that of natural rape root exudate was pumped through the membrane at 0.5 ml/h. Scanning electron microscopic examinations indicated that the inoculum formed microcolonies embedded in exopolymers evenly distributed over the membrane surface. The lipid composition and content of poly-beta-hydroxybutyrate in free-living and adhered cells were determined by gas chromatography. The bacterial consumption of amino acids in the exudate was also studied.     

Model system for studies of microbial dynamics at exuding surfaces such as the rhizosphere.

An autoclavable all-glass system for studying microbial dynamics at permeable surfaces is described. Standard hydrophobic or hydrophilic membranes (46-mm diameter) of various pore sizes were supported on a glass frit through which nutrient solutions were pumped by a peristaltic pump. The pump provided a precisely controlled flow at speeds of 0.5 to 500 ml of defined or natural cell exudates per h, which passed through the membrane into a receiving vessel. The construction allowed a choice of membranes, which could be modified. The system was tested with a bacterium, isolated from rape plant roots (Brassica napus L.), that was inoculated on a hydrophilic membrane filter and allowed to develop into a biofilm. A defined medium with a composition resembling that of natural rape root exudate was pumped through the membrane at 0.5 ml/h. Scanning electron microscopic examinations indicated that the inoculum formed microcolonies embedded in exopolymers evenly distributed over the membrane surface. The lipid composition and content of poly-beta-hydroxybutyrate in free-living and adhered cells were determined by gas chromatography. The bacterial consumption of amino acids in the exudate was also studied.     

Columnar organization of the optic tectum in the frog

Cobaltous lysine complex was used to label tectal cells. Cobalt soaked into a piece of filter paper and placed onto the surface of the tectum labelled neurons in the whole thickness of the tectum below the filter paper. The labelled area was sharply demarcated from the unlabelled tectal tissue. Focal cobalt injections into different tectal layers labelled small groups of cells and the cobalt-filled structures were perpendicularly oriented to the surface of the tectum. Efferent axons could be followed into layer 7, but other lateral connections were very sparse. These results support the hypothesis that the tectum has columnar organization similar to that of the mammalian neocortex.     

Incorporation of P and Growth of Pseudomonad UP-2 on n-Tetracosane

Cultures of the marine pseudomonad UP-2 growing on n-tetracosane contained both free cells and cells bound to the solid hydrocarbon. After separation by filtration through a Whatman no. 1 filter, the numbers of free and bound cells were estimated from the amount of P incorporated into each fraction and the determined value of P incorporation per viable cell in the filtrate (free cells). During the early exponential growth phase, over 80% of the cells were bound to large pieces of n-tetracosane; as the culture approached the stationary phase, the number of bound cells remained constant, whereas free cells continued to accumulate. Pulse-labeling experiments indicated that cells grew both on the surface of the solid and in the aqueous medium. During the growth cycle, a portion of the n-tetracosane which was initially nonfilterable was recovered in the filtrate in a form which was largely cell associated. This cell-associated n-tetracosane was preferentially utilized and could completely account for the observed growth of free cells.     

Direct quantitation and detection of salmonellae in biological samples without enrichment, using two-step filtration and real-time PCR

A new two-step filtration protocol followed by a real-time PCR assay based on SYBR green I detection was developed to directly quantitate salmonellae in two types of biological samples: i.e., chicken rinse and spent irrigation water. Four prefiltration filters, one type of final filter, and six protocols for recovery of salmonellae from the final filter were evaluated to identify an effective filtration protocol. This method was then combined with a real-time PCR assay based on detection of the invA gene. The best results were obtained by subsequent filtration of 100 ml of chicken rinse or 100 ml of spent irrigation water through filters with pore diameters of >40 mum to remove large particles and of 0.22 microm to recover the Salmonella cells. After this, the Salmonella cells were removed from the filter by vortexing in 1 ml of physiological saline, and this sample was then subjected to real-time quantitative PCR. The whole procedure could be completed within 3 h from sampling to quantitation, and cell numbers as low as 7.5 x 10(2) CFU per 100-ml sample could be quantified. Below this limit, qualitative detection of concentrations as low as 2.2 CFU/100 ml sample was possible on occasion. This study has contributed to the development of a simple, rapid, and reliable method for quantitation of salmonellae in food without the need for sample enrichment or DNA extraction.     

Frequency Dependent Topological Patterns of Resting-State Brain Networks

The topological organization underlying brain networks has been extensively investigated using resting-state fMRI, focusing on the low frequency band from 0.01 to 0.1 Hz. However, the frequency specificities regarding the corresponding brain networks remain largely unclear. In the current study, a data-driven method named complementary ensemble empirical mode decomposition (CEEMD) was introduced to separate the time series of each voxel into several intrinsic oscillation rhythms with distinct frequency bands. Our data indicated that the whole brain BOLD signals could be automatically divided into five specific frequency bands. After applying the CEEMD method, the topological patterns of these five temporally correlated networks were analyzed. The results showed that global topological properties, including the network weighted degree, network efficiency, mean characteristic path length and clustering coefficient, were observed to be most prominent in the ultra-low frequency bands from 0 to 0.015 Hz. Moreover, the saliency of small-world architecture demonstrated frequency-density dependency. Compared to the empirical mode decomposition method (EMD), CEEMD could effectively eliminate the mode-mixing effects. Additionally, the robustness of CEEMD was validated by the similar results derived from a split-half analysis and a conventional frequency division method using the rectangular window band-pass filter. Our findings suggest that CEEMD is a more effective method for extracting the intrinsic oscillation rhythms embedded in the BOLD signals than EMD. The application of CEEMD in fMRI data analysis will provide in-depth insight in investigations of frequency specific topological patterns of the dynamic brain networks.     

Ultrastructure of the protonephridia of larval Rugiloricus cf. cauliculus, male Armorloricus elegans, and female Nanaloricus mysticus (Loricifera)

The protonephridial system of several Loricifera was studied by transmission electron microscopy. A larval specimen of Rugiloricus cf. cauliculus possesses two protonephridia, which are "capped" frontally by a compact mass of still undifferentiated gonadal cells. Each protonephridium consists of four monociliary terminal cells and four canal cells with a diplosome but no cilia. Because of incomplete series of sections and unsatisfactory fixation, the outleading cell(s) could not be detected. In a male specimen of Armorloricus elegans, each gonad contains two protonephridia that open into the gonadal lumen. Each protonephridium consists of two monociliary terminal cells, each forming a filter, two nonciliated canal cells, and two nephroporus cells. The protonephridial lumina of the latter cells fuse to one common lumen, which unites with the gonadal lumen. Preliminary observations on the protonephridia of a female Nanaloricus mysticus reveal a more complicated arrangement of interdigitating terminal and canal cells. One or two terminal cells form their own individual filter or four cells form a common compound filter. The cilium of the terminal cells of all species investigated are surrounded by a palisade of nine microvilli that support the filter barrier made of an extracellular matrix. An additional filter diaphragm could be traced between the pores in the cell wall of each terminal cell of A. elegans. The urogenital system of the Loricifera differs from that of the Priapulida in that the protonephridia of the former are completely integrated into the gonad, whereas the excretory organs of the latter open into the urogenital duct caudally of the gonads.     

Observation of fibronectin distribution on the cell undersurface using immunogold scanning electron microscopy.

Immunogold staining followed by observation with scanning electron microscopy (SEM) has been quite effective in showing the distribution of proteins on dorsal cell surfaces. However, observation of proteins on the ventral cell surface using SEM has not been developed to the same extent. In this study, human gingival fibroblasts cultured on titanium-coated wafers were embedded in resin. After fracturing the wafers off the embedded cells, the undersurface of the cell was exposed by argon gas glow discharge etching. After 15 min of glow discharge etching, the resin covering the cell undersurface was completely removed. The distribution of fibronectin (FN) on the cell undersurface was demonstrated using an anti-FN antibody and colloidal gold (30 nm) conjugated with IgG. The undersurface was then coated with carbon or gold-palladium and observed by SEM. Using backscattered electron detection, gold beads could be identified in high contrast. On cells cultured for 5 hr, gold beads were distributed randomly on the entire cell undersurface. However, a line of gold beads was sometimes observed close to the edge of the cell. These results indicated that this immunogold/SEM etching method provides a powerful means for studying cell adhesion molecules on the cell undersurface. (J Histochem Cytochem 47:1487-1493, 1999)     

MMPI Drug-Eluting IVC Filter Decreases Adhesion Between Caval Wall and Filter

The implantation of inferior vena cava (IVC) filter was a safe and effective therapy for preventing fatal pulmonary embolism. However, there are risks associated with long-term implantation of filters. Retrievable filters are designed to be removed, but may also remain permanently. Retrieval can reduce risk of long-term complications. The difficulty or impossibility of retrieval is still an issue of retrieval filter. The major causes of filters retrieval failure were intimal overgrowth and severely tilted filter with apex embedded into the caval wall. Matrix metalloproteinases (MMPs) play a key role in neointimal hyperplasia. It is documented that neointimal hyperplasia can be reduced by inhibiting MMP activity and hence smooth muscle cell migration. MMP inhibitors (MMPI) can potently inhibit the activity of MMPs. We hypothesize that a drug-eluting filter which contains MMPI may inhibit IVC neointimal hyperplasia and decrease the adhesion between vascular wall and filter struts. After implantation of drug-eluting retrieval filter, MMPI is released slowly at the sites where the filter struts are in contact with the caval wall; the activity of MMPs of caval wall will be inhibited, injury in basement membrane is decreased, migration of SMC maybe reduced, and the release of extracellular matrix maybe lessened. Finally, neointimal hyperplasia maybe inhibited, the adhesion between vascular wall and filter maybe weakened, the success rate maybe increased, and the vascular injury during retrieval maybe reduced. The hypothesis might improve the long-term prognosis of venous thromboembolism patients.     

A membrane filter direct count technique for enumeratingBdellovibrio

A membrane filter direct count method was devised for enumeratingBdellovibrio cells in “clean” suspensions. The procedure involves filtering a specified volume of a diluted, Trisbuffered suspension ofBdellovibrio cells through a known area of a 100-nm-pore-size Millipore brand membrane filter. A clarification solvent was used to render the filter transparent, so that the bdeloyvibrios on the filter could be photomicrographed and counted either visually or by means of a Quantimet 720 Image Analyzing Computer. The number ofBdellovibrio cells per milliliter in the undituted suspension could be calculated from the mean number of cells per unit area of the filter, the dilution factor, and the volume of diluted suspension filtered. TheBdellovibrio cells were distributed on the filters in a Poisson manner when there were not more than about 3.5 cells per 100 μm² of filter surface. The membrane filter direct counts correlated well with direct counts obtained by the Petroff-Hausser method. The correlation of direct counts with plaque (“viable”) counts showed that 80 to 95% of the direct-countedBdellovibrio cells in the clean suspensions were capable of forming plaques on lawns of suitable substrate bacteria. *** DIRECT SUPPORT *** A01R4002 00007     

FSH-initiated differentiation of newt spermatogonia to primary spermatocytes in germ-somatic cell reaggregates cultured within a collagen matrix

We previously cultured fragments of newt testes in chemically defined media and showed that mammalian follicle-stimulating hormone (FSH) stimulates proliferation of spermatogonia as well as their differentiation into primary spermatocytes (Ji et al., 1992; Abe and Ji, 1994). Next, we indicated in cultures composed of spermatogonia and somatic cells (mainly Sertoli cells) that FSH stimulates germ cell proliferation via Sertoli cells (Maekawa et al., 1995). However, the spermatogonia did not differentiate into primary spermatocytes, but instead died. In the present study, we embedded large reaggregates of spermatogonia and somatic cells (mainly Sertoli cells) within a collagen matrix and cultured the reaggregates on a filter that floated on chemically defined media containing FSH; in this revised culture system, spermatogonia proliferated and differentiated into primary spermatocytes. The viability and percentage of germ cells differentiating into primary spermatocytes were proportional to the percentage of somatic cells in the culture, indicating that differentiation of spermatogonia into primary spermatocytes is mediated by Sertoli cells.     

Mouse 3T3 cell filtrability correlating with concanavalin A agglutinability

In a previous study of the dextran gel sphere model system, a possible correlation between cell deformability and agglutinability by concanavalin A was indicated. Cell deformability was evaluated as filtrability, using polycarbonate membrane filtration. With 25-mm diameter filters and 5-ml cell suspensions at (0.8–16) · 10⁵ cells/ml, the filtrability at a given filter pore size was highly reproducible and was not affected by variations in cell population, viability, washings of cells retained on filter, or temperature. The filtrability of EDTA-dissociated 3T3 cells through 12-μm pore size filter was 8%, and a suspension of 10⁶ cells/ml was not agglutinated by 600 μg concanavalin A. The filtrability of trypsin-dissociated 3T3 cells was 95%, and these cells were agglutinated by 200 μg of the lectin. EDTA-dissociated SV-3T3 cells had a filtrability of 73% and were also highly agglutinable. Formalin fixation reduced the high filtrability to 6%, and also abolished the agglutinability. As a further test of the correlation, trypsin-dissociated 3T3 cells were admixed with the fixed cells. The agglutinability varied with the proportions of the two cell components, and the admixtures could be separated according to filtrability into the original components with distinctly different agglutinability. Furthermore, 25% of a random population of EDTA-dissociated SV-3T3 cells retained by the filter were found to be non-agglutinable. The separated SV-3T3 cell fractions could also form admixtures of different agglutinability. It is concluded that the agglutinability of mouse 3T3 and SV-3T3 cells by concanavalin A can be correlated with the predicted by cell filtrability.