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Immunohistochemical signal amplification by catalyzed reporter deposition and its application in double immunostaining.

The biotinyl-tyramide substrate of the horseradish peroxidase enzyme has been recently introduced to amplify immunohistochemical signals. We applied either fluorochromeor biotin-conjugated tyramine to improve the detection of different antigens in sections of rat stomach, pancreas, and hypothalamus. A ten- to 100-fold increase in staining efficiency was achieved, depending on the antibody, with either fluorescent or peroxidase detection systems. The amplification method was particularly useful for increasing a weak signal of conventional immunostaining caused by suboptimal tissue fixation. At a very low concentration of the primary antibody, the antigen can no longer be detected by a conventional fluorescent secondary antibody but is still detectable after amplification. When an antibody is used at this very low concentration and is detected by a fluorescent amplification method, another primary antibody, raised in the same host species, can be used and demonstrated with a different fluorochrome in subsequent conventional immunostaining of the same section. In this way it becomes possible to immunostain the same section with two different primary antibodies raised in the same host species. Samples for such double immunostaining are demonstrated here using pairs of monoclonal antibodies (to tyrosine hydroxylase and oxytocin) in the hypothalamus and polyclonal antibodies (to glucagon and neurofilament M) in sections of rat pancreas. Because in many cases the availability of antibodies is limited, the amplification method can be a quick and efficient tool for double immunostaining with antibodies from the same host species.

Animals↗

Effect of the dural application of Zenker's solution on the feline brain.

Zenker's solution is a tissue fixative containing mercuric chloride, potassium bichromate, sodium sulfate, and glacial acetic acid. In 1956, Anderson and Johnson reported its use in clinical neurosurgery. They applied the solution to the exposed dura after craniectomy. Delayed bone formation was thought to be due to the suppression of the osteoblastic activity of the outer layer of the dura. The fixative has since become a well-accepted adjuvant to the treatment of craniosynostosis. In 1972, Pawl and Sugar reported postoperative seizures in 6 of 34 patients treated with this solution. They assumed that the fixative penetrated the dura and irritated or damaged the cortex. To clarify the effect of Zenker's solution on the underlying brain, we performed bilateral parasagittal craniectomies in a group of kittens and adult cats. Zenker's solution was applied to one side and the other side served as a control. The animals were killed after periods varying from 24 hours to 2 months. We then examined the cortex under the craniectomies. There was immediate breakdown of the blood-brain barrier, as evidenced by the penetration of intravenous Evans blue. In the postoperative period investigated, an inflammatory response in the underlying brain with thickening of the arachnoid occurred. The results and implications of these experiments are presented. (Neurosurgery, 6: 45--48, 1980)

Acetates↗

Ouabain binding during plasma membrane biogenesis in duck salt gland.

The conditions necessary for optimal ouabain binding in the avian salt gland were examined. Binding was enhanced by ATP and Mg2+ and was decreased by K+, but was unaffected by added Na+. Both maximal binding and complete inhibition of Na, K-ATPase activity were obtained at 1 X 10(-6) M ouabain. Half maximal binding and half maximal inhibition of Na, K-ATPase activity were obtained at 1.7 X 10(-7) M ouabain. Ouabain binding increased in parallel with increasing specific activity of the Na, K-ATPase duringsalt-induced salt gland specialization. The ratio of Na, K-ATPase activity to ouabain-binding sites remained constant during the salt stress as well as after removal of the salt diet. Autoradiography indicated binding to partially and fully differentiated secretory cells of the salt gland. The ouabain binding assay appeared to be a more useful indicator of membrane amplification than Na, K-ATPase activity since it is rapid, essentially irreversible, less sensitive to tissue fixatives, and quantitatively measured the number of enzyme molecules.

Adenosine Triphosphatases↗

Experimental applications of porous (coralline) hydroxylapatite in middle ear and mastoid reconstruction.

Hydroxylapatite is a ceramic implant material which has been extensively investigated in the orthopedic and oral surgical literature. A set of animal experiments was carried out to determine the feasibility of the use of porous (coralline) hydroxylapatite for middle ear and mastoid reconstruction. Coralline hydroxylapatite was placed in the mastoid bulla of dogs and was carved into struts which were placed into the middle ear and against the tympanic membrane. Coralline hydroxylapatite is a highly biocompatible material which showed abundant ingrowth when in contact with host bone. There was also good soft-tissue ingrowth and fibrous tissue fixation within the middle ear and against the tympanic membrane. Hydroxylapatite is generally accepted to be osteoconductive, but not osteogenic. However, one of the specimens described with chronic inflammatory mucosal changes, shows de novo new bone formation within the pores of the graft. While normally the bone formed in coralline hydroxylapatite is secondary (lamellar), in this specimen primary (woven) bone was found. Porous (coralline) hydroxylapatite appears to be an excellent otologic graft material. It biointegrates with the host, is osteoconductive, and under certain circumstances may be osteogenic.

Animals↗

Distal axonopathy: one common type of neurotoxic lesion.

Neurotoxic chemicals commonly produce retrograde degeneration of the axons of long and large nerve fibers in the central and peripheral nervous system. This produces a clinical picture of polyneuropathy in man and animals in which sensory and motor disturbances develop in the feet and hands then progress with time to the legs and arms. Distal axonopathy, as the underlying pathologic process is termed, is one of four principal types of neurotoxic diseases, the others including degeneration of neurons (neuronopathy), myelin sheaths (myelinopathy) and damage to the neurovasculature (neurovasculopathy). In the experimental animal, these four types of neurotoxic diseases can be distinguished by examining selected areas of brain and nerve tissues prepared by contemporary methods of tissue fixation. These procedures may form the basis of a new and sensitive assay for neurotoxicity.

Aging↗

In vivo analysis of trophoblast cell invasion in the human.

In vivo analysis of trophoblast cell invasion is highly dependent on histological techniques, which are amply described in standard textbooks. The emphasis of this chapter therefore lies on material collection and interpretation of tissue sections, rather than on histological techniques per se. Proper identification of vascular structures on placental bed histological sections is important, the more because invading trophoblastic cells induce significant structural changes in uterine blood vessels, which may be disturbed in complicated pregnancies. Guidelines for distinguishing several vascular structures are provided, and different approaches for qualitative and quantitative assessment of spiral artery changes are discussed. The purpose of such studies is not only to obtain a better insight into mechanisms of trophoblast invasion and associated maternal tissue changes, but also to understand placental bed defects in various pregnancy complications.

Arteries↗

Current opinion on computer-aided surgical navigation and robotics: role in the treatment of sports-related injuries.

Computer-assisted surgery (CAS) may allow surgeons to be more precise and minimally invasive, in addition to being an excellent research tool. Medical imaging, such as magnetic resonance and computed tomography is not only an important diagnostic tool, but also a necessary planning tool. In orthopaedic sports medicine, precision is needed when placing tunnels for soft tissue fixation of replacement grafts. Two types of CAS systems -- passive and active -- have been developed. Passive systems, or surgical navigation systems, provide the surgeon with additional information prior to and during the surgical procedure (in real time). Active systems have the ability of performing certain surgical steps autonomously. Both active and passive CAS systems are currently a subject of basic science and clinical investigations and will be discussed and commented on in this article. In summary, passive navigation systems can provide additional information to the surgeon and can therefore lead to more precise tunnel placement. Active robotic technology seems to be accurate and feasible with promising initial results from Europe. However, active and passive CAS can only be as precise as the surgeon who plans the procedure. Therefore, future studies have to focus on integrating, arthroscopy, 3-D image-enhanced computer navigation, and virtual kinematics, as well as to increase precision in surgical techniques.

Athletic Injuries↗

Nail cosmetics: allergies and irritations.

Recent precise figures are not available for the number of adverse reactions related to the use of nail care products. Reactions to nail cosmetic procedures may be divided into reactions at the site of application to the nail itself and ectopic reactions, when the hand transfers a small amount of nail cosmetic to other areas of the skin. Fingernail coatings encompass two types: coatings that harden upon evaporation (nail enamel, base coat, top coat); and coatings that polymerize (sculptured nails, light-curing gels, preformed artificial nails, nail mending and nail wrapping). The test battery enables us to distinguish allergic reactions from irritant reactions. Interestingly, some reactions, such as distant allergic contact dermatitis, are more frequent with nail enamel than with coatings that polymerize. On the other hand, the latter are greater offenders in the nail area. Nail hardeners may just be modified nail enamels containing nylon fibers, acrylate resin and hydrolyzed proteins. Others may contain up to 5% formaldehyde tissue fixative (which can have adverse effects on the nail), but are designed in the US to be applied only to the free edge of the nail while the skin is shielded. Caution is necessary in interpreting formaldehyde patch testing reactions.

Acrylates↗

Microdissection and the study of cancer pathways.

Abstract: The study of genetic alterations in tumors and their precursor lesions is often hampered by the presence of a heterogeneous background of non-neoplastic elements such as stromal cells, inflammatory cells, and angiogenic elements. Microdissection involves the extraction of specific populations of cells under direct visualization. In this article, we will discuss the currently available techniques of microdissection, and briefly review how this material is being utilized in the study of cancer pathways. Microdissected tissue is amenable for the study of cancer genomics, expression analysis and most recently, cancer proteomics. The purity of reagents obtained from microdissected material has resulted in the successful identification of tumor suppressor genes as well as novel transcripts and proteins that are altered in neoplastic cells. Improved techniques of tissue fixation and microdissection, supplemented with ancillary technology such as pre-amplification, have permitted the use of increasingly smaller quantities of material for the study of cancer pathways. Importantly, it is now possible to analyze many of the genetic changes that precede cancer, thereby identifying populations "at risk" for developing malignancies in the future.

Cell Separation↗

Immunolabeling with the protein A-gold technique: an overview.

The protein A-gold technique is a postembedding immunolabeling method that permits light- and electron-microscopic localization of intracellular antigens on tissue sections with high sensitivity and resolution. The article reviews practical aspects of tissue fixation and embedding protocols as well as methodologic aspects of the protein A-gold technique; careful control of these interrelated factors is the prerequisite for successful application. The technique is demonstrated by a few selected examples of application in basic research and in applied medical research. The protein A-gold technique provides a good alternative to existing techniques for immunolabeling in diagnostic pathology.

Animals↗

Formaldehyde prepared from paraformaldehyde is stable.

For critical histological investigations, tissue fixation is sometimes carried out in formaldehyde freshly prepared from paraformaldehyde by heating. The purity of formaldehyde produced in this way is superior to that of commercial stock solutions. We studied the stability of freshly prepared formaldehyde solutions by determination of pH and titration of acid, which reflect the formation of formic acid. It was found that very small amounts of acid are produced during the heating of paraformaldehyde. Prolonged heating or storage of freshly prepared formaldehyde for up to 8 days did not significantly increase the amount of acid. It was also found that heating of the paraformaldehyde is not necessary, since depolymerization may take place at room temperature. We conclude that formaldehyde prepared from paraformaldehyde remains stable for considerable periods of time, and it is therefore unnecessary to prepare it immediately prior to fixation. Also, in many cases, buffering of the fixative may be omitted, since only minor changes in the pH occur during fixation.

Animals↗

Histologic and microradiographic evaluation of textured and nontextured aluminum oxide dental implants.

This paper reports the histological and microradiographical observations of macroscopically textured and nontextured aluminum oxide dental implants. Ion beam bombardment was used to produce a wafflelike pattern of surface undulations approximately 35 microns deep. Twelve nontextured and eight textured implants were placed in the healed extraction sites of the second and forth mandibular premolars of adult mongrel dogs. Eight nontextured and five textured implants remained in situ for six months. Results consistent with previously reported clinical and radiographic findings, indicated a decreased incidence of an interposed fibrous tissue-implant interface associated with the textured implants. Texturing was not found to influence the amount of vertical bone loss. Secondarily, delayed tissue fixation due to post-retrieval mechanical testing procedures resulted in loss of valuable cellular information.

Aluminum↗

Ultrastructural study of the liver in argininosuccinase deficiency.

The liver of a 19-year-old woman with argininosuccinase deficiency was studied ultrastructurally under conditions for excellent tissue fixation. Dilated smooth endoplasmic reticulum and mitochondrial crystalloids were the most prominent abnormalities found. These features were compared to fine-structural abnormalities found in other disorders of the Krebs-Henseleit pathway. No change was considered specific or related to subcellular enzyme localization.

Adult↗

[The mechanism of positive scintigraphy with 99mTc-pertechnetate in adenolymphomas of the parotid grand].

In order to clarify the mechanism of positive scintigraphy with Tc pertechnetate in adenolymphomas of the parotid gland, 10 cases of adenolymphoma were studied morphologically and from a functional view point. Salivary tissue obtained from normal parotid glands were also investigated for comparison with the tumor. In morphological studies, rapid tissue fixation by microwave irradiation produced very good results for examining mitochondria by high resolution light microscopy. A number of mitochondria were found both in the epithelial cells of adenolymphoma and in the striated duct cells of the parotid gland. In functional studies, the one-step method of enzymatic histochemistry was used for detecting Na-K ATPase. Na-K ATPase was found on the baso-lateral side of the epithelial cells of adenolymphoma and the striated duct cells of the parotid gland, and this activity of Na-K ATPase may enhance the accumulation of Tc pertechnetate in these cells. These observations may provide evidence for the bi-cellular theory which indicates that adenolymphoma originated from immature intercalated duct reserve cells.

Adenolymphoma↗

Improved power control during microwave heating in biological applications.

The heating performance of prototype variable power control units attached to two domestic microwave ovens, 500 W and 700 W, was investigated. The units employed the phase control method where rated voltage was obtained by switching AC supply at a particular angular position on the sinusoidal voltage. In order to create experimental conditions employed in a previous study, Sydney tap water (50 mL) was heated at a 10% power level in the 500 W oven. Water boiled at 10 min in both experiments. However, the precision and control of the heating was greatly improved with addition of the control unit. A disinfecting solution (50 mL) and a tissue fixative (10 mL) were included for experiments with the 700 W oven. A power level of only 1% was sufficient to maintain the temperature of the fixative at low temperatures (30-40 degrees C) where many biological reactions occur. The present results indicate that microwave heating power can be controlled by the variable power control method. This will make temperature control possible through the provision of an electronic feedback loop which links a thermocouple with the power control unit.

Heating↗