Preparation tissue

Plant tissue preparation procedures for calorimetric analyses are often very simple excise a section of plant tissue and place it in the calorimeter ampule. However, some standard precautions must be observed and some tissues require special handling or treatment to ensure meaningful measurements. 

Microbial contamination is a universal concern. In most studies, microbial metabolic activity on plant tissue surfaces contributes negligibly to overall metabolic activity and can be ignored. Contamination in plant cell cultures and plant tissues that have been severely damaged may be significant, however, and should be carefully considered. Microbial infection within tissue sections may have a significant effect on measured heat rate, but this has only been studied in a few tissues [29, 30]. Usually, the presence of microbial contamination can be detected by the characteristic exponential increase in heat rates with time characteristic of microbial growth. 

Addition of buffer or nutrient solution to plant tissue samples in the calorimeter ampule are generally not needed. In closed ampules, the vapor phase is rapidly saturated with water in the presence or absence of added buffers. Water loss from the tissues to the head space is small and insignificant with larger tissue samples. With very small samples compared to ampule volume, the water loss from the tissue to achieve saturation may cause significant tissue dehydration so water should be added to the ampule, but preferably not in contact with the sample. Dipping leaf or meristem tissue in water just prior to calorimetric measurement or 

Exceptions to the practice of not adding buffer to plant tissue samples are numerous. Root tissues must be maintained moist and consequently are examined while in close contact with buffer-saturated filter paper disks [34, 35]. Immersing root tissue in water does not yield satisfactory data. Callus tissue cells and cell cultures are commonly measured while being maintained on agar or in liquid media. Marine tissues may be examined in water suspension by flow calorimetry, but unstirred samples rapidly settle to the bottom of the ampule and become O2 limited during batch calorimetry. 

An immediate problem that must be overcome when conducting an RRA is the relatively low density of receptors specific for the ligand vis-a-vis other tissue constituents that are available for nonspecific interactions. Indeed, in many competitive binding assays, the amount of ligand specifically bound when all receptors are occupied (saturated) is on the order of fmoles/mg protein Because m most applications, and particularly for RRAs, non- 

For many receptor binding assays, tissue may be stored frozen without an appreciable loss of receptor number or receptor 

In summary, preparation of tissues serves to enhance both the precision and the sensitivity of the RRA technique. Although general similarities in preparative procedures exist among assays, each RRA has been tailored to a specific purpose and the techniques established dunng the course of developing the assay should be adhered to with rigor 

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Differences in the equilibrium dissociation constant, K, for the binding of the various saxitoxins to the sodium channel binding site largely determine the differences in the potencies of the toxins in whole animal assays and in tissue preparations. 

Excitable tissue preparations were obtained fresh daily from live animals using the technique described by Dodd et al. (12). Protein was measured on each synapto-some preparation using the Coomassie Brilliant Blue dye technique described by Bradford (13) results were expressed as "toxin bound per mg synaptosome protein". 

Research in this area advanced in the 1970 s as several groups reported the isolation of potent toxins from P. brevis cell cultures (2-7). To date, the structures of at least eight active neurotoxins have been elucidated (PbTx-1 through PbTx-8) (8). Early studies of toxic fractions indicated diverse pathophysiological effects in vivo as well as in a number of nerve and muscle tissue preparations (reviewed in 9-11). The site of action of two major brevetoxins, PbTx-2 and PbTx-3, has been shown to be the voltage-sensitive sodium channel (8,12). These compounds bind to a specific receptor site on the channel complex where they cause persistent activation, increased Na flux, and subsequent depolarization of excitable cells at resting... 

Because of their strategic localization, astrocytes play a crucial role in maintaining the extracellular ionic homeostasis, provide energetic metabolites to neurons and remove excess of neurotransmitter in schedule with synaptic activity. In addition, the strategic location of astrocytes allows them to carefully monitor and control the level of synaptic activity. Indeed, number of papers during the last 15 years have shown that cultured astrocytes can respond to a variety of neurotransmitters with a variety of different patterns of intracellular calcium increases (Verkhratsky et al. 1998). Later on, studies performed in intact tissue preparations (acute brain slices) further established that the plasma membrane receptors can sense external inputs (such as the spillover of neurotransmitters during intense synaptic activity) and transduce them as intracellular calcium elevations, mostly via release of calcium from internal stores (Dani et al. 1992 Murphy et al. 1993 Porter and McCarthy... 

Compound NPYR Species Rat Tissue Preparation Liver microsomes Comments identification of 4-hydroxybutyr-aldehyde (as its 2,4-dinitrophenyl-hydrazone) from a-hydroxylation Reference 9... 

Compound Species Tissue Preparation Comments Reference... 

Ki of 6 nM against a guinea-pig uterine oxytocin preparation and at least 100-fold selectivity over rat vasopressin tissue preparations. These molecules show a pseudo-irreversible pharmacology, with an extended action against spontaneous contractibility of rat uterus 24 h post partum. 

Displacement binding experiments with 3H-PCP were conducted with minor modifications of the filtration method described in the literature (Zukin and Zukin 1979 Vincent et al. 1979 Hampton et al. 1982) in crude tissue preparations of rat whole brain homogenates in 5 mM Tris-HCl (pH 7.4). 

Pertwee RG, Fernardo SR, Griffin G, Abadji Y, Makriyannis A. Effect of phenylmethylsulphonyl fluoride on the potency of anandamide as an inhibitor of electrically evoked contractions in two isolated tissue preparations. Eur J Pharmacol 1995b 272 73-78. 

The effects of D-glucose observed in vivo are not well reproduced in vitro. Madara [203] reported that cytoskeletal contraction and enhanced paracellular permeability were observed only in an in situ perfusion preparation and not in an isolated tissue preparation. Although its in vivo effect was not tested, 25 mM D-glucose, an effective concentration in the jejunum [47], failed to enhance the in vitro transport of sotalol (log PC = -0.62), atenolol (log PC = 0.16), or nadolol (log PC = 0.93) across the isolated conjunctiva [213], For a similar reason and possibly due to the absence of a Na+-glucose cotransporter in the cornea, 25 mM D-glucose was ineffective in increasing the corneal transport of these three drugs. 

As a gold standard, fresh tissue prepared by snap-frozen method, cut by cryostat, and fixed in acetone, ethanol, or other non-cross-linking fixatives, has been generally accepted as reliable. 

Two different and possibly complementary approaches have been explored. One utilizes a panel of quantifiable internal reference standards (QIRS), which are common proteins present widely in tissues in relatively consistent amounts.11,22 In this instance because the reference proteins are intrinsic to the tissue they are necessarily subjected to identical fixation and processing, and incur no additional handling or cost, other than synchronous performance of a second IHC assay (stain), such that the intensity of reaction for the QIRS and the test analyte can be compared by IA, allowing calculation of the amount of test analyte (protein) present on a formulaic standard curve basis. The other approach seeks to identify external reference materials and to introduce these into each step of tissue preparation for cases where IHC studies are anticipated in this instance the logistical issues of production, distribution, and inclusion of the reference standard into all phases of tissue processing also must be considered, along with attendant costs. 

Leong3 postulated that internal controls were required to optimize the variable influences resulting from aspects of tissue preparation and factors intrinsic to the staining method. Various controls have been employed, includ-... 

Williams JH, Mepham BL, Wright DH. Tissue preparation for immunocytochem-istry. /. Clin. Pathol. 1997 50 422-428. 

Principle Biosensors consist of paper matrixes and tissue enzyme preparations, often the pure enzyme AChE or AChE-containing cells. As seen in Fig.l, main scheme of the preparation technology and procedure includes (i) the preparation of same kinds of biotests-biosensors, which are paper matrixes impregnated with tissue preparation of AChE and covered by polymer film. (ii) biochemical reactions of the AChE activity with and without inhibitors tested and (iii) the photometric analysis of the samples for quantitative estimation of the biochemical reactions. 

A variety of different types of tissue preparation are used to study neurosecretion and synaptic transmission. A classical preparation is the frog NMJ (discussed below). The brain slice has been used for many years for biochemical studies of CNS metabolism and is a useful preparation for electrophysiological studies of synaptic transmission in the CNS. Slices can be oriented to maintain the local neuronal circuitry and can be thin, 0.3 mm, to minimize anoxia. The transverse hippocampal slice is widely used as an electrophysiological preparation to study synaptic plasticity (see Ch. 53). Primary cultures of neurons from selected CNS areas and sympathetic ganglia are also frequently used. They permit excellent visual identification of individual neurons and control of the extracellular milieu, but the normal neuronal connections are disrupted. 

Immunocytochemical methods have been widely applied to visualize proteins, carbohydrates, or lipids in sectioned material. The advantage of using immunocytochemistry is to be able to localize the molecules of interest within the tissue. Several procedures have been described. Basically, these procedures can be split into four main steps that are described in subheadings (1) tissue preparation, (2) the primary antibodies, (3) the visualization of the target, and (4) enhancement of signals with antibody complexes. In addition, a protocol for alkaline phosphatase will be presented in detail in Subheading 5. The terms primary and secondary antibodies refer to the order in which they are applied to the target. The immunocytochemical procedures are not limited to sectioned... 

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