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  • 1990-1994  (9)
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  • 1
    ISSN: 1573-4838
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine , Technology
    Notes: Glutaraldehyde (GA) crosslinking (fixation) of collageneous tissues is a widely used method for the preparation of implantible tissues to be used as biomaterials. In an attempt to optimize the fixation process, experiments were carried out with two types of collagen (native collagen membrane and synthetic collagen sheet) to study the effect on crosslinking of temperature, GA concentration and fixation time. Secondly, stimulation of GA diffusion was studied and finally, a procedure of low T-presoaking followed by brief exposure to high temperatures was investigated. As a measure of the degree of crosslinking the shrinkage temperature (T s) was determined. Temperature (20°C or 45°C), concentration (0.1% or 1.0%) or fixation time (4 or 24 h) were found to be positively correlated with the T s of the collagen sheets. Whereas untanned collagen exhibits a T s of around 60°C, short-term (1 or 5 min), high-temperature (50°C) fixation with a 0.1% GA solution caused the shrinkage temperature to increase to 72°C and 85.1°C, respectively. Fixation with 0.01% GA for 5 min at 50°C appeared equally effective as 1 min with 0.1% GA (T s=70°C). Microwave irradiation showed to be slightly more effective in enhancing the crosslinking process compared with conventional heating. Surprisingly, at any combination of temperature, concentration and fixation periods of 4 h or 24 h, an increased T s towards the central regions of the collagen was observed. Soaking the samples at 20°C (1 h) or at 0°C (3 h) with subsequent short-time heating to 45°C caused an almost equal rise in T s throughout the collagen samples and is therefore recommended for preparing implantable tissues.
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  • 2
    Electronic Resource
    Electronic Resource
    Springer
    Journal of molecular histology 22 (1990), S. 381-388 
    ISSN: 1573-6865
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary Microwave technology has become important in preparatory techniques for microscopy in many different ways. This paper discusses various aspects of the physics of microwaves. It gives some theoretical background to understand the practical procedures. Some peculiarities in the optics of microwaves are pointed out, and the practical implications in particular of choosing the size and shape of samples and containers are discussed. Diffusion rates and chemical-reaction rates increase exponentially with temperature, so that precise temperature control is essential in most histochemical procedures. Such control is complicated by localized heating of the system, and of temperature sensors themselves, which may occur as a result of microwave irradiation.
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  • 3
    ISSN: 1573-6865
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary The effect of short time heating of aqueous solutions of glutaraldehyde (GA) on relative aldehyde concentration was determined using spectrophotometric analysis. Because free monomeric GA absorbs U. V. light at 280 nm, whereas the alpha, beta polymeric forms absorb at 235 nm, the purity of GA solutions can be expressed as the ratio: A 235 nm/A 280 nm (purification index, P.I.). Heating of 4 ml aliquots of 0.85% distilled aqueous GA solution resulted in an increase of the absorption at 280 nm which is correlated positively with temperature. No increase of absorption at 235 nm was found when solutions were kept at 40°C for several hours. The increase of absorption at 280 nm is caused by a rapid decyclization of hemiacetals producing an increase in free aldehyde concentration. No major differences in absorption were found between the solutions heated by microwave and by conventional heating. However, because microwave irradiation is known to produce an homogeneous rise in temperature, especially in bulky samples, it is expected that the results of fixation procedures will improve by the combined effect of higher temperature and enhanced diffusion rates of the fixating species.
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  • 4
    ISSN: 1573-6865
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary A DNA-in situ hybridization protocol was adapted for application to sections of routinely processed paraffin embedded material. This protocol was developed previously for detecting DNA-virus infected cells in whole cell preparations and employs biotinylated DNA as probe. Three different biotin detection methods were optimized and applied. The first uses streptavidin and a biotinylated complex of alkaline phosphatase, the second consists of an immunogold-silver staining, and the third of a peroxidase technique using a silver amplification. The alkaline phosphatase method was the most rapid, and as sensitive as the immunogold-silver staining. The peroxidase method was the most sensitive. Microwave irradiation was applied to the different incubation steps of these three detection methods. Short incubations with microwave irradiation gave very poor results when peroxidase labelled antibodies were used. Short incubation with microwave irradiation gave results comparable to those obtained with conventional incubations, when streptavidin, antibiotin, complexed alkaline phosphatase, or gold labelled goat antirabbit were used. It was thus shown that microwave irradiation creates the possibility of a very rapid label-detection for nonradioactive DNA-in situ hybridization.
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  • 5
    Electronic Resource
    Electronic Resource
    Springer
    Journal of molecular histology 22 (1990), S. 335-340 
    ISSN: 1573-6865
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary A morphological and morphometrical study was performed on testicular cells after microwave stabilization of the tissue while immersed in phosphate buffered saline (PBS), 0.9 NaCl or Tris-HCl. Fixation in Carnoy's fluid without irradiation was chosen as a control chemical fixation method. After microwave stabilization or chemical fixation, the testes were embedded in paraffin or in plastic (glycolmethacrylate). An excellent morphology, comparable to that after chemical fixation in Carnoy's fluid, was observed in the plastic sections of tissue irradiated in PBS or NaCl, even when the sections were subsequently treated with an aggressive reagent at high temperature, required for the Feulgen reaction. The nuclear area of the microwave-stabilized Sertoli cells was 37–46% smaller in haematoxylin-eosin stained, paraffin sections in comparison with that in the glycolmethacrylate sections. The microwave-stabilized, paraffin-embedded tissue was much more vulnerable to the hot HCl treatment of the Feulgen staining than the chemically fixed tissue, resulting in an additional 10–20% decrease in nuclear size. The latter finding is particularly important for quantitative microscopy, where the Feulgen staining method is often employed.
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  • 6
    Electronic Resource
    Electronic Resource
    Springer
    Journal of molecular histology 22 (1990), S. 341-346 
    ISSN: 1573-6865
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary In this article two applications of microwaves in histopathology, microwave-stimulated staining of tissue sections and microwave-stimulated fixation of cryostat sections, are reviewed. For a good understanding of the influence of microwaves on physico-chemical processes like staining and fixation the relevant physics are included. Major advantages of microwave techniques are speed and/or improved quality. The cryostat-microwave technique appears to be well-suited for the demonstration of intermediate filament proteins: the sensitivity of monoclonal antibodies directed against keratins and vimentin can be substantially increased using the ethanol based fixative Kryofix.
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  • 7
    Electronic Resource
    Electronic Resource
    Springer
    Journal of molecular histology 22 (1990), S. 347-352 
    ISSN: 1573-6865
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary The Streptavidin-Biotin Complex (SABC) Immunostaining method can be carried out by performing the rinsing and blocking steps and in addition the incubations with the primary and the secondary antibody sera in the microwave oven. Irradiation of the Streptavidin-Biotin Complex reduces stain activity due to destruction of horseradish peroxidase (HRP) (Boon & Kok, 1988). Therefore, we decided not to perform this step in the microwave oven. The microwave incubations can be performed using antisera dilutions of 1:1000 (instead of 1:50) in tissue fixed with Kryofix, allowing staining in staining racks. This keeps hands-on time low and simplifies the microwave steps. It is very difficult to obtain reproducible results in the microwave oven using droplet incubations due to problems with hot spots and antenna effects. These problems are avoided when cuvettes are used and air is blown through the solutions during microwave incubations. With effective temperature control the method is highly reproducible, and takes at least 100 min less than the conventional SABC method. It is, in particular, attractive when large series of slides must be routinely immunostained and when reproducible results are desired.
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  • 8
    ISSN: 1573-6865
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary The fixation of the neurotransmitter dopamine in the central nervous system by perfusion with formalin solutions seems to take place mainly via the formalin-induced condensation product norsalsolinol. In the present investigation the influence of microwave irradiation of the formalin-induced condensation of dopamine was studiedin vitro andin vivo by making use of different, relatively low, formalin concentrations. It appeared thatin vitro andin vivo the dopamine conversion was complete with 4% formalin and no influence of microwaves was noted. However, by making use of much lower formalin concentrations (0.2% and 0.4%) the condensation of dopamine was strongly augmented,in vitro (200%) andin vivo (at least 500%) using microwave techniques. There was a considerable loss in non-microwaved tissue (30%) after perfusionin vivo. This was lower (10%) in microwaved tissue. In experiments with perfused brain tissue which allowed a more complete calculation, a loss was found. This might be caused by a strong binding of dopamine and/or norsalsolinol to tissue components or to side reactions that could not be traced by the present experimental techniques.
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  • 9
    Electronic Resource
    Electronic Resource
    Hoboken, NJ : Wiley-Blackwell
    Journal of Biomedical Materials Research 28 (1994), S. 1013-1025 
    ISSN: 0021-9304
    Keywords: Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine , Technology
    Notes: Porcine collagen membranes having a rough and a smooth side were used for subcutaneous implantation studies in rats. Two tanning protocols were used for the membranes, a new one involving microwaving and glutaraldehyde treatment (NEWGA), and the other, a conventional method using glutaraldehyde treatment at room temperature (OLDGA). Untreated membranes (NONGA) were also implanted. Sections of the implants were examined by light microscopy and with the confocal laser scanning microscope focusing on neovascularization and incorporation of the implant. At 64 days, the smooth sides of NEWGA and OLDGA implants were not well incorporated, with scarring subjacent to the surface and dystrophic calcification of that side of the membrane. At the same time, the rough sides of the NEWGA and OLDGA were not calcified with a giant cell reaction around the porcine collagen. The best incorporation was found in the NONGA membranes with no dystrophic calcification, excellent neovascularization of all layers, and complete remodeling at day 64. After 5 months, the completely remodeled NONGA membrane still could be identified, and the NEWGA and OLDGA membranes were calcified with a giant cell reaction having a dense fibrous capsule. It is concluded that if cross-linking is deemed necessary, the microwave cross-linking method is advisable because in the early stages there is less reactive inflammation around it, and the implant surfaces should be rough with an open structure, making calcification of crosslinked collagen unlikely. © 1994 John Wiley & Sons, Inc.
    Additional Material: 16 Ill.
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