Histochemical technique

A variety of different methodologies has been employed to investigate the neurotransmitter systems involved in control of behavioral states. Biochemical experiments have elucidated the pathways and enzymes involved in the synthesis, degradation, release and reuptake of different neurotransmitters. Immuno-histochemical techniques have allowed the visualization of their cellular and sub-cellular distribution throughout the nervous system as well as the distribution of their receptors and uptake systems. Chemical sampling techniques, including... 

The detection of serotonin in nervous and non-nervous tissue was aided by the development of the Falck-Hillarp histochemical technique, a method whereby freeze-dried sections of tissue, when exposed to formaldehyde vapour cause indoleamines to emit a yellow fluorescence. Dahlstrom and Fuxe used this technique to show that the highest concentration of serotonin in the brain is located in the raphe nuclei, projections from these cell bodies ascending to the forebrain via the medial forebrain bundle. Descending fibres were also shown to project to the dorsal and lateral horns and the intermediolateral column of the spinal cord. Detailed observation of the distribution of the serotonergic system in the brain became possible with... 

By the use of histochemical techniques and immunoelectrophoresis, Nielsen and Bjerrum (1977) identified four major protein complexes in milk lipid globule membrane. They found that xanthine oxidase was located on the internal face of the membrane and that the other three complexes, as well as Mg2+-adenosine triphosphatase and 5 -nucleo-tidase, were accessible on the outer surface of the membrane. Based on available information, it cannot be determined if any proteins of the globule membrane span the lipid bilayer, as do certain proteins of other membranes (Singer 1974 Rothman and Lenard 1977). 

The avidin-biotin complex (ABC) method and the streptavidin-biotin (SAB) method are more sensitive than the peroxidase-antiperoxidase (PAP) method for histochemical techniques. The strong noncovalent attraction between biotin and avidin or streptavidin is exploited in many histochemical, immunohistochemical, and in situ hybridization... 

The avidin-biotin system was developed for detecting antigens at the electron microscope level (Heitzmann and Richards, 1974). Later Heggeness and Ash (1977) proposed the use of this system for fluorescence immunohistochemistry. Guesdon et al. (1979) proposed a variety of labeled avidin-biotin methods which were further supported by Warnke and Levy (1980). The avidin-biotin methods used today are similar to the system described by Hsu et al. (1981). This system is a significant improvement over the previous immuno-histochemical techniques. The problem of endogenous biotin is discussed on page 98. 

Conjugation of enzymes to antigens or antibodies were first developed for histochemical techniques and were used for localization of antigens and antibodies in tissue sections (60). 

Effects on the liver of rats exposed to chromium compounds have been detected by biochemical and histochemical techniques. Rats treated by gavage with 13.5 mg chromium(VI)/kg/day as potassium chromate for 20 days had increased accumulations of lipids (Kumar and Rana 1982) and changes and... 

Culling, C. F. A. Handbook of Histopathological and Histochemical Techniques, Buttaworths, London 1974... 

Histochemical techniques for detecting substrate before and after enzyme treatment are extremely useful in studies on cellular structure. One of the oldest histochemical tests utilized saliva to identify suspected glycogen or starch. More definitive results are obtained when thin sections of a tissue are incubated in a buffered solution of purified amylase and stained for poly-utc-glycols. Material stained by periodic acid-Schiff reagent in the control, but not in the section exposed to amylase, is assumed to be glycogen or starch. Two more of the numerous histochemical techniques associated with localization of substrate are—using hya-luronidase to locate hyaluronic acid and chondroitin 4- and 6-sulfates (179) and using neuraminidases to locate sialomucins (180). By use of electron microscopy in combination with the histochemical technique subcellular localization can be obtained. 

When histochemical techniques for acid phosphatase were carried out at the electron microscopic level (N5, N6), dense bodies about 0.4 /t in diameter having a single outer membrane were observed in intact cells of the liver. In addition bodies with similar morphological characteristics were found to be located along the fine bile canaliculi. Indeed lysosomes as a whole showed considerable polymorphism, apparently the result of their association with the different materials that are phagocytized by the cell (S29). 

Microscopic, electron-microscopic, and histochemical techniques would help in studying the actual state of carbohydrates in soils. 

Palacios, G., A double immunocytochemical and histochemical technique for demonstration of cholinergic neurons and microglial cells in basal forebrain and neostriatum of the rat, Neurosci. Lett., 115, 13, 1990. 

In addition to the epidermal abnormalities noted above, there is an increase in the number of dermal cells which is probably of secondaiy importance. However, it should be noted that Weddell (W7, W8) using special histochemical techniques has reported an increased production and breakdown of Schwann cells in the psoriatic lesion, and he believes that these changes are of primary importance in stimulating the epidermal lesion. Along similar lines is the suggestion by Pinkus and Mehregan (P7) that the polymorphonuclear leukocytes are of prime importance in stimulating the epidermis and that the dilated capillary in the papilla squirts these leukocytes into the epidermis as an initial event. 

If no other histochemical techniques are needed, fresh tissue can be used. The block must be frozen quickly without cracking. Tissue is cut in small blocks with a new razor blade (washed in soap to remove oil). An alternative to the method below is freezing tissue on aluminum foil on top of dry ice and surrounding with crushed dry ice. However, the perimeter of the tissue is usually poorly preserved with this method. For RNA detection, DEPC is included at 0.02% in the subbing and fixation solutions. Poly-L-lysine coating may be superior at temperatures > 50°C since gel from gel-coated slides may detach at higher hybridization temperatures. It is convenient to use only some of the slides for immediate hybridization and store the rest for future reference. 

Clark EM, White JL Jr., Patterson PM. Improved histochemical techniques for the detection of Acremonium coenophialum in tall fescue and methods of in vitro culture of the fungus. J Microbiol Methods 1 149-155, 1983. 

The data on other transmitter candidates in the hippocampus are less complete. All parts of the hippocampal region contain noradrenaline (NA) terminals which are, however, diffusely distributed relative to ChAc and constitute but a small proportion of the afferents (Blackstad et al., 1967). The highest levels are found in hilus of area dentata and in stratum radiatum of regio inferior. The distribution of nerve terminals containing serotonin (5-HT) has not been described, apart from stating that it is different from that of NA terminals (Fuxe et al., 1970), probably because of the instability of the fluorescence obtained with this substance in the histochemical technique. However, there has been a report of 5-HT terminals on the pyramidal cell bodies in the cat (Eidelberg et al., 1967). 

With the rapid increase in understanding of the mechanisms of cell injury and repair, a number of new substances have been identified that may prove to be useful markers of acute injury or disease activity. These include various cytokines and growth factors, several lipid mediators, a complex array of extracellular matrix components and cell adhesion molecules, plus a variety of miscellaneous compounds. At the present time, the clinical utility of their measurement in biologic samples is unknown, although in selected instances, clinical correlates have emerged. Unfortunately, not all of these markers are present in urine or blood samples. For some, detection involves histologic or histochemical techniques applied to renal tissue samples. Nonetheless, the substances discussed below are intimately involved in the control and modification of cell function, the response to stress and/or the processes of repair. It is anticipated that with proper amplification, one or more may be useful as a marker of susceptibility, exposure or effect. 

The ability to quantify mRNA biochemically and semiquantitatively, by histochemical techniques, has led to a number of studies examining the factors involved in their regulation within the brain. Most workers have concentrated on the hippocampus, as this area contains the highest concentration of each mRNA , overcoming some of the technical difficulties associated with the detection and quantification of the very low levels seen in other regions. Initial experiments have used procedures, such as induction of seizures, that alter electrical activity, since it is well known that ab-... 

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