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The use of gold reagents to quantitate antibodies eluted from nitrocellulose blots: application to electron microscopic immunocytochemistry.

An assay is described in which gold reagents were used to quantitate nanogram amounts of antibody that had been eluted from antigens immobilized on nitrocellulose paper. Standard curves were generated by the application of rabbit immunoglobulin G (IgG) to nitrocellulose sheets assembled in a dot blot matrix apparatus. Blots were stained using either colloidal gold or immunogold, enabling quantitation of IgG concentration by scanning densitometry. Linear and reproducible standard curves were obtained. As little as 1 ng IgG/dot could be quantified using either gold reagent. In contrast to colloidal gold, immunogold could be used specifically to quantitate rabbit IgG regardless of the presence of bovine serum albumin or antigen coeluted from the nitrocellulose blot. The applicability of the immunogold assay was demonstrated by fractionating a complex rabbit antiserum raised against the RIM protein of frog retinal rod outer segments. Anti-RIM antibody was affinity-purified, quantitated by the immunogold assay, and subsequently employed in immunocytochemical studies using thin sections of retina embedded in a hydrophilic plastic, LR-Gold.

Animals↗

Cytokinetic and morphological changes in the lungs and lung-associated lymph nodes of rats after inhalation of fly ash.

Fischer-344 rats (male and female) were exposed to 36 mg/m3 of fluidized bed coal combustion fly ash or sham-exposed for 7 hr/day, 5 days/week for 4 weeks, and sacrificed after 2 or 4 weeks of exposure and at 2, 22, and 42 weeks after the end of exposure. Animals were injected with tritiated thymidine 2 hr before sacrifice and autoradiographs prepared from 1-micron sections of lung and lymph node tissue embedded in glycol methacrylate plastic. Differences in labeling indices of pulmonary epithelial cells, alveolar macrophages, airway epithelial cells, and cells of the lung-associated lymph nodes between the exposed and control animals were maximal after 2 and 4 weeks of exposure. Labeling indices for lung epithelial cells were about the same in control and exposed animals at 2, 22, and 42 weeks after the end of exposure. However, these values were elevated relative to earlier control levels. In contrast, morphological changes in the fly ash-exposed animals were most prominent after the end of the exposure. These changes included thickening of the alveolar walls, clusters of particle-filled macrophages in the alveolar region, and perivascular inflammation. Additionally, there were small granulomas in the alveolar region at 42 weeks after the end of exposure. Granulomas were also formed in the lung-associated lymph nodes and and bronchus-associated lymphoid tissue. We conclude that the inhalation of fly ash alone had little detrimental effect upon the rat lung. However, the increases in proliferation indicate the potential for fly ash combined with a carcinogen to enhance the carcinogen's effect.

Animals↗

A study of the odontoblast process with transmission electron microscopy.

Freshly extracted human third molars with completely formed roots were sectioned and placed in Karnovsky's fixative. The specimens were decalcified in ethylenediaminetetraacetic acid (EDTA) for 91 days, followed by digestion in collagenase. They were then fixed in a solution of buffered osmium tetroxide or ruthenium red and buffered osmium tetroxide, embedded in Spurr's plastic mixture, and sectioned for transmission electron microscopy. Each section was stained with uranyl acetate and lead citrate and viewed with a transmission electron microscope. No odontoblast processes could be identified at the cemental third of the dentin with the described technique.

Adult↗

Local fluorouracil chemotherapy interferes with neural and behavioral recovery after brain tumor-like mass compression.

In this study, we investigated the impact of intracerebral delivery of chemotherapy on functional recovery from focal cortical tissue displacement, characteristic of brain tumors. Unilateral focal brain compression was induced by epidural implantation of an inverted hemisphere-shaped bead over the sensorimotor cortex. Microinjections of a total of 1mg chemoagent fluorouracil or the same volume of saline were made into the compressed cortex. Behavioral tests of forelimb sensorimotor function were conducted during 4 weeks' observation. Rats subjected to any of the three types of lesions, saline microinjection plus cortical compression, chemoagent microinjection alone, or chemoagent microinjection combined with cortical compression, demonstrated significant behavioral deficits in several sensorimotor tasks, compared with saline-microinjected control animals. In placing tests, behavioral deficits elicited by each single treatment were worsened by combined treatment with chemoagent microinjection and focal cortical compression. Concurrently, local delivery of chemoagent into the compressed cortex induced increased cortical tissue loss, necrosis and apoptosis. These data indicate that local chemotherapy exacerbates compression-induced neurological impairment, and a model of controlled focal cortical compression may provide a valuable means to improve anti-cancer therapeutic designs with reduced deterioration of brain function.

Animals↗

Ganciclovir intraocular implant. A clinicopathologic study.

BACKGROUND: Surgical implantation of the intraocular sustained-release ganciclovir device is a safe and effective treatment for cytomegalovirus (CMV) retinitis. Previous histopathologic studies on eyes containing such implants have been limited by the necessity of removing the device before processing. Microtome sectioning of hard plastics within paraffin-embedded blocks is infeasible, and the anatomic relations of implant to eye are destroyed. METHODS: The authors studied four eyes from three patients who had undergone implant insertion. Globes with implants in place were fixed in neutral 10% formation, embedded in methylmethacrylate, sectioned on a special microtome, and stained with hematoxylin-eosin. RESULTS: After methacrylate embedding, the precise anatomic relations of the implant to the neighboring uveoscleral coats were preserved. In two eyes, the suture tab of the implant protruded through the sclera, exiting subconjunctivally. In two eyes, the implant was totally intravitreal. In all patients, the device was supported by fibrous tissue which emanated from a surgical coloboma of the pars plana ciliaris. Focal granulomatous inflammation adjoined suture and implant materials but no other inflammation or deleterious effects on the ocular structures were noted. CONCLUSION: This report is the first to document the intraocular histopathology of the ganciclovir implant. The subconjunctival location, enhancing the potential for endophthalmitis, may be avoided by trimming of the suture tab close to the anchoring suture and not tying it too tightly. Methylmethacrylate embedding is a useful technique for preserving the microanatomy of intraocular implants.

AIDS-Related Opportunistic Infections↗

Technical report: Cell thickness measurements by confocal fluorescence microscopy on C3H10T1/2 and V79 cells.

Measurements of C3H10T1/2 and V79 cell thickness were performed on living cells by confocal laser fluorescence microscopy. Thickness distributions are reported for cells growing as a monolayer (on mylar and glass) and suspended in their medium. Mean values for cells grown on mylar (corrected for refractive index effects) are 2.9 +/- 0.6 and 6.1 +/- 1.0 microm for C3H10T1/2 and V79 cells respectively. Mean values of the diameters of cells suspended in their medium are 13.0 +/- 1.6 and 9.3 +/- 1.4 microm for C3H10T1/2 and V79 respectively. Knowledge of cell thickness, as irradiated, is of central relevance for studying the relative biological effectiveness of low energy, poorly penetrating radiations. It can be concluded, from the measured cell thickness distributions, that with C3H10T1/2 cells grown on mylar, the LET variation through the whole cell is within 20% for protons and alpha-particles with energies down to 0.6 and 2.5 MeV respectively. From a comparison with thickness values reported in the literature for living or fixed embedded cells growing on plastic substrate, mean values between 2.4 and 3.4 microm and between 6 and 7.5 microm could be assumed for C3H10T1/2 cells and for the most widely used V79 cell lines respectively.

Animals↗

Differential effects of protease digestion on photoreceptor lectin binding sites.

The use of lectin cytochemistry together with proteolytic digestion techniques to partially characterize lectin binding sites of several intracellular compartments in frog photoreceptors was studied. Uniform access of reagents to all intracellular compartments was obtained by performing the experiments directly on semithin sections of retinal tissue embedded in a hydrophilic plastic resin. Protease pretreatment of sections of Xenopus laevis eyecup leads to a loss of wheat germ agglutinin (WGA) binding sites from most of the rod outer segment. Under experimental conditions used here, cone outer segment WGA binding sites are resistant to proteolytic digestion. Another major difference between rod and cone under segments is that rod outer segments are heavily labeled with succinylated WGA, whereas cone outer segments are barely labeled except for a region of intense staining thought to be at the connecting cilium. WGA binding sites in the shed outer segment tip (phagosome) are also relatively resistant to proteolytic digestion, as is the tip region of a few rod outer segments. This difference in lectin binding properties between the bulk of the outer segment membrane and the shed outer segment membrane is the only distinction we have observed between the two compartments in terms of their glycoconjugates. These results may be useful in terms of designing experiments to isolate cone and rod outer segments separately. They indicate that a change in outer segment glycoconjugates may accompany the shedding and phagocytosis events, as previously suggested, but this change does not necessarily involve the addition of saccharides to outer segment glycoproteins.

Animals↗

Health smart cards: merging technology and medical information.

Smart cards are credit card-sized plastic cards, with an embedded dime-sized Integrated Circuit microprocessor chip. Smart cards can be used for keyless entry, electronic medical records, etc. Health smart cards have been in limited use since 1982 in Europe and the United States, and several barriers including lack of infrastructure, low consumer confidence, competing standards, and cost continue to be addressed.

Confidentiality↗

Identification of normal or neoplastic murine mesenchymal cells in chimeric tissues or heterotransplants.

An improved method for identifying murine mesenchymal cells in chimeric tissues or heterotransplants using Hoechst dye 33258 is described. Following fixation in formalin-saline, tissues are embedded in JB-4 plastic. Sections 3 micron thick are then stained in a 10 microgram/ml solution of Hoechst 33258 in Hanks' balanced salt solution for 5-10 min at 4 C. After rising, the sections are coverslipped using a modified polyvinyl alcohol mounting medium. This approach offers several advantages over existing techniques: 1) uniform section thickness is more easily obtained than with paraffin or cryostat microtomy, thereby allowing improved resolution and more reliable identification of mesenchymal cells with small nuclei such as skeletal muscle myocytes or fibroblasts, 2) the preparations are stable over long periods and can be repeatedly viewed or photographed, and 3) calcified tissues can be examined without prior decalcification. An example is shown of species identification using rat chondrosarcoma cells grown in nude mice.

Animals↗

Preload loss in gold prosthesis-retaining screws as a function of time.

PURPOSE: Screwed joints are widely used in implant dentistry, and their failure is a significant problem that may be related to loss of preload. Preload is the compressive force generated across a joint when a screw is tightened and is responsible for keeping a joint closed. For a given torque, preload is limited by the frictional resistance of the contacting screw threads, flange, and opposing joint surfaces. This study tested the hypothesis that following correct placement, prosthetic gold screws lose preload over time. MATERIALS AND METHODS: The study used standard Nobel Biocare components. Strain gauges mounted on a standard abutment formed a transducer to measure preload. Five sets of new prosthetic gold screws, gold cylinders, and standard abutment screws were assembled in turn on the top of an implant body, using the transducer abutment. The gold screws were tightened with a Nobel Biocare Torque Controller set to 10 Ncm, when it had an output of 12.06 +/- 0.8 Ncm with 95% repeatability. Preload was monitored for 15 hours; then the screws were removed and examined under a scanning electron microscope. RESULTS: Preload ranged from 157.5 to 488.9 N (mean 319.6 N), with a mean reduction over 15 hours of 24.9%, with 40.2% of this occurring within 10 seconds of tightening. DISCUSSION: Torsional relaxation of the screw shaft, embedment relaxation, and localized plastic deformation of the gold alloy and opposing titanium threads were the most likely explanation for this phenomenon. CONCLUSIONS: New prosthetic gold screws suffer significant loss of preload following placement.

Compressive Strength↗

Monitoring of visual function during parasellar surgery.

Damage to the visual system is an unfortunate complication of surgery in the area of the optic chiasm. It is now possible tomonitor the functional status of the visual system intreoperatively at regulat intervals. This is accomplished by recording the Visual Evoked Response to flashes of light from light-emitting-eiodes. These diodes are embedded in a special plastic shell which inserts under the eye lids of each eye. Since the light comes from the diodes in the plastic shell, there is no need to disturb the surgical procedure when a test run is desired. A record is obtained by averaging 100 three-per-second flashes.

Adenoma, Acidophil↗

Embedding method for electron microscopy in biology.

Transmission electron microscopes were commercially available in some foreign countries in the later 1930s. Sectioning for light microscopy was used for electron microscopic observations of the internal structure of cells and tissues in the early days. Paraffin waxes were tested as embedding media, but they were too soft to enable thin sections. Pease and Baker (1948) (74) achieved an early success with the double-embedding method using the plastic "Parlodion" and paraffin wax.

Epoxy Resins↗

A revision of the Spalteholz method for visualizing blood vessels.

A transparent embedding method is described which enables blood vessels to be visualized in the organs of experimental animals. The circulatory system is perfused with a mixture of fixatives, latices and a pigment. The tissue is embedded in a solid plastic with an appropriate refractory index. The method allows the scoring of blood vessel abnormalities in thick sections. It may also be of use for quantitative histology and the localization of electrodes in thick sections.

Animals↗

Introduction to smart card technology and initial medical application.

Smart card technology is the name applied to the use of a plastic card with an embedded computer chip. Recent development of smart card software has allowed storage and retrieval of medical information, affording the opportunity to provide a standardized, portable, accessible medical record for use in prehospital and emergency department patient encounters. We describe the smart card concept and its initial deployment in a section of a large Midwestern urban area.

Emergencies↗

Multiple labeling in electron microscopy: its application in cardiovascular research.

The heart is a muscular pump kept together by a network of extracellular matrix components. An increase in collagens, as in chronic congestive heart failure (CHF), is thought to have a negative effect on cardiac compliance and, thus, on the clinical condition. Conventional electron microscopy allows for the study of cellular and extracellular components and scanning electron microscopy (SEM) can put these structures in three-dimensional perspective. However, in order to study extracellular matrix components in relation to cells, immunoelectron microscopy is superior. We have used this technique in our studies on heart failure. Heart specimens were fixed in 4% paraformaldehyde and 0.1% glutaraldehyde in sodium cacodylate buffer, dehydrated by the method of progressive lowering of temperature and embedded in LR Gold plastic. Immunolabeling could be achieved with different sized gold-conjugated secondary antibodies or protein-A gold conjugates. Depending on the objective, ultra small gold (USG) conjugates or a regular probe size can be used. Labeling efficiency could be increased by bridging antibodies. The double and triple staining procedures were based on single staining methods using one- and two-face labeling. The choice of antibodies and gold conjugates depended on the objectives. Immunoelectron microscopy, using multiple labeling, allowed a detailed study of the organization of the extracellular matrix and its relationship with cardiac myocytes. This may prove to be a useful tool for the study of chronic heart failure.

Actins↗

The influence of the embedding medium when staining for electron microscopy: the penetration of stains into plastic sections.

Using a sectioned-section procedure it was found that only a few structures, e.g. the connective tissue elements, were deeply penetrated by phosphotungstic acid (PTA) and uranyl acetate (UA). Reynold's lead citrate appeared to penetrate more generally. Using stains selective for the embedding medium, and also observing the surfaces of shadowed sections, it was concluded that structures readily penetrated by polar stains were only poorly infiltrated by non-polar embedding media. It is argued that this differential infiltration leading to differential penetration by polar stains largely controls the pattern of staining of ultrathin sections by PTA and UA.

Animals↗