Experimental procedure specimen preparation

In this chapter more detailed information on the double-cell voltage-clamp setup and protocols for assessing gap junctional conductivity is given, as well as a description of the cell-isolation procedure for this purpose and cell culture models. Information on immunocytochemical localization of gap junctions and on the experimental procedure of preparing specimens and slides for immunohistology is given. A protocol for isolation of gap junction proteins is also outlined. Readers interested in more details of the cell-culture technique regarding incubators, sterile technique, etc., and different isolation and culture protocols are referred to more specialized literature [Lindl and Bauer, 1994 Piper, 1990]. 

Before applying the vacuum microbalance or any similar method to the study of the rate of a particular surface reaction it is essential to understand as much as possible concerning the chemistry of the main reaction and the possible side reactions which may occur in a given system. This requires detailed thermochemical calculations to be made for all conceivable reactions to determine the specifications for the vacuum system and furnace tubes, the preparation of the specimens, the experimental procedures, and the interpretation of the data obtained. Kinetic theory calculations should be applied to aid in interpretation of the rates of certain vacuum and low-pressure reactions. 

Results from the initial resin studied are also being employed in the development of additional experimental procedures. Plans are currently being drafted to prepare three-ply test specimens that are similar to the specimens used in the initial study, with the middle ply consisting of solid polystyrene. Comparing specimens with and without the graft polymers introduced to the ply interfaces should provide additional information on the ability of the cellulosic graft polymers to facilitate bonding between wood and plastic materials. If this approach proves successful, additional procedures will then be developed for the production of simple composite specimens. 

Properties and reactivity of a calcium sulfate 3-hemihydrate are widely dependant on the preparation procedure. Although kinetical studies on the hydration reaction are done by a number of researchers, there is no standard procedure to elaborate a stable, reproducible calcium -hemihydrate specimen. This paper presents a very simple apparatus and an experimental procedure setup to elaborate a specimen that is stable and allows kinetical studies even after a long time of conservation. This apparatus also allows the simultaneous determination of the hydration rate for six different species of incompletely hydrated plasters and demonstrates experimentally that the amount of heat involved in the hydration... 

For species characterization, whole-cell and extracellular proteins (CUnk and Pennington 1987 Berber et al. 2(X)3) or cellular fatty acid analysis and/or combined with gas chromatography may be applied. In general, fatty acid profiles correlate with the data on DNA homology and phenotype features (Behme et al. 1996). Recently, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDl-TOF MS) was experimentally applied to intact bacterial cells or specimens prepared nsing extraction procedures. This chemotaxonomic method was used for identification of the staphylococcal species and MRSA strains (Du et al. 2(X)2 Carbonnelle et al. 2007). 

This chapter was written to provide abrief discussion of instrumentation, present the advantages and disadvantages of specimen preparation techniques for biochemical research, assess the sources of error and artifact, provide selected examples of applications, and outline general experimental procedures, where they can be prescribed appropriately. 

Specimen preparation requires patience, practice, and meticulous attention to cleanliness and detail. While there are many specimen preparation techniques that exist, six will be discussed here, as they are the most useful for biochemical research. These techniques are sufficiently complex and multifaceted that a separate chapter could be written on each. In some cases, specific procedures must be empirically derived for each type of specimen and experimental condition. For these, we refer the reader to excellent reviews of a particular subset of specimen preparation procedures or to specific papers that contain a well-developed methods section. For more generic procedures, we provide a general description of the protocol. 

Cryosectioning. For some antigens in very low titer, the above techniques may not be successful. Cryoultrathin sectioning, developed by Tokuyasu (60), followed by labeling, may be the method of choice. This method is perhaps the most technically complex and difficult of all TEM specimen preparation techniques. A well-frozen sample is a necessity and this method also requires an ultramicrotome with the cryounit attachment. The specifics of this technique and experimental procedures have been discussed in great detail in two reviews (60, 61). 

Standard objects of known size are the simplest calibration tools. If an internal calibration is possible, it is often better than an external standard. For example, a common procedure is to measure latex particle diameters in the TEM. Particles with very accurately known diameters (and a narrow size distribution) are readily available. Some of these particles can be added to the sample suspension before preparation for microscopy. They are used as internal standards to correct for any changes there might be either during specimen preparation or during observation in the microscope. They must not be so close in size to the experimental particles that they may be confused, or so different that the same image magnification would not be suitable ... 

When the PEELS measurement was conducted, an abrupt drop in density was observed at the interface between the matrix and the craze bands (Figure 4). In addition, a drop of approximately 50% in density was found at the base of the already unloaded craze band. This observation implies that an extension ratio of at least 2 exists for the craze fibrils. This phenomenon is not uncommon for thermoplastic crazes (5, 10). To ensure that the PEELS method gives reasonable results, the density of the craze band inside a polystyrene tensile specimen was measured (Figure 5) using the same sample-preparation procedures described in the section Experimental Details. The measured density of the craze band in the unloaded polystyrene was found to be about 0.62 g/cm3, which is in good agreement with the number reported in the literature (5,10, 24). 

The first triaxial tests on deep-sea sediment cores were performed by Moore (1964). These were run on samples from the experimental Mohole off Mexico and were mostly from sub-bottom depths greater than 75 m. Sample preparation for these compact specimens would not have been much different from conventional land procedures. 

Successful application of this experimental approach depends on several factors synthesis of high-quality hybridization probes, appropriate fixation of the sample, the hybridization procedure, and the fluorescence microscopy approach used to image the specimen. In adapting the technique of three-dimensional in situ hybridization to different organisms and tissue types, the simplest and most invariant aspect of the technology has proved to be the hybridization procedure. Probes must be developed on a custom basis to address the particular questions of the investigator, and equally crucially, fixation conditions need to be adapted with special attention to the physical attributes of the individual specimen. However, once appropriate preparation conditions are established for a particular type of sample, it has been unnecessary to reoptimize the basic hybridization protocol. We discuss each of these experimental issues separately below. 

The remaining part of this chapter will review the three most common direct methods for measuring fiber-matrix adhesion, focusing on the sample preparation and fabrication, the experimental protocols and the underlying theoretical analyses upon which evaluation of these methods are based. In addition, finite-element nonlinear analyses and photoelastic analyses will be used to identify differences in the state of stress that is induced in each specimen model of the three different techniques. In order to provide an objective comparison between the three different techniques to measure the interfacial shear strength for the prospective user, data and a carbon fiber-epoxy resin system will be used as a baseline system throughout this chapter, However, these methods and procedures can be applied for adhesion measurements to any fiber-matrix combination. 

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