Silver Staining of Insect Central Nervous Systems by the Bodian Protargol Method

Improved fixation of ganglia of the central nervous system of Periplaneta americana and Schistocerca gregaria for silver staining by Power's (1943) modification of the Bodian protargol method is given by alcoholic Bouin aged for at least 40 days at 60° C. During impregnation of sections, increased copper and decreased pH give paler staining, more selective for nerve fibres. Prolonging impregnation from 24 to 48 hours weakens the stain and decreases selectivity. The intensity of the stain depends chiefly upon the amount of unreduced (developable) silver combined with the tissues; selectivity is determined mainly by the number and distribution of the reduced silver particles (‘nuclei’). In development, increased sodium sulphite gives more differentiation, increased hydroquinone gives less. Optimum developer composition depends upon impregnation, and thick sections need more differentiation than thinner ones. Within limits, change in one of the factors that control staining can be balanced by changes in others, but by suitable adjustment of the conditions the result can be varied from almost total staining of nerve fibres, for general neuroanatomy, to highly selective staining for tracing individual fibres.     

Combination of non-specific cholinesterase histochemistry and immunofluorescence staining for the study of the sensory innervation of skin and muscle

Summary In the present study we describe the application of the non-specific cholinesterase (nChE) histochemical method for the detection of encapsulated sensory nerve endings prior to immunofluorescence staining of the sensory nerve fibres. The nChE staining of Schwann-derived structures surrounding sensory terminals allowed us to identify unequivocally the sensory corpuscles in the skin and the muscle proprioceptors (muscle spindles and Golgi tendon organs) in longitudinal sections of muscle tissue. The nChE staining of sensory nerve endings and immunofluorescence-labelled nerve fibres and their terminals could be viewed and photographed in the same section using appropriate filters. Since nChE activity persists in terminal Schwann cells for a long time after loss of the sensory axons, this combined enzyme- and immunohistochemical approach is also useful for experimental studies involving denervation and re-innervation of sensory nerve endings.     

Ammoniacal silver method in alkaline pH range as a technique for detection of morpho-functional features of nerve tissue elements]

An alkaline range pH 9.1-12.3 instead of values pH 7.0-7.1 was used to localize lysine-rich and arginine-rich nucleoproteins by ammonia silver (AS) staining method (Black and Ansley, 1966) in brain structures of rabbits and rats. This modification of AS method made it steady to reveal lysine-rich and arginine-rich nucleoproteins and was able to demonstrate the high variability of protein localization in relation to the balance of essential amino acids in the brain structures with different functions. The best results of morphological staining of nerve, glial cells and their fibres were found at pH 10.6--the point of maximal dissociation of lysine--NH2 groups. At the same time the new modification of AS method allows the use of all alkaline range pH 9.1-12.3 for revealing the cells of nervous tissue and to vary the treatment of sections with silver ions.     

Simple Fluorescence Staining of Insect Central Nerve Fibres with Procion Yellow

Nerve fibres in the central nervous system of the cockroach Periplaneta ameri-cana can be displayed by staining whole ganglia for 1-2 hr in a saturated solution of Procion yellow M-4R in cockroach saline diluted to maintain isotonicity. Selected fibres are stained preferentially by cutting a peripheral nerve or interganglionic connective close to the ganglion, or damaging neuron cell-bodies. The ganglion is washed in saline, fixed in alcoholic Bouin, dehydrated and embedded. Under fluorescence microscopy, sections show stained fibres brilliant yellow against a green background. The method is simpler than intracellular injection and demonstrates even the finest fibres.     

Ultrastructural visualization of elastic fibres with a tannate-metal salt method

Summary A modification of the tannic acid-metal salt method was applied as an ultrastructural stain for elastin. Thin sections of glutaraldehyde-fixed, embedded rat aorta and rabbit elastic cartilage, with and without osmication, were examined. Raising the pH of the tannic acid solution from 2.7 to 9.0 progressively increased the electron-density of elastic fibres and collagen fibrils in osmicated and unosmicated specimens. The maximum tannic acid staining of elastic fibres was observed in the pH range 7.0–9.0. Collagen staining, although less intense than that of elastic fibres, was also greatest in this pH range. Elastic fibres in osmicated specimens demonstrated the strongest tannic acid staining with a minimal increase in density of collagen and cell nuclei when compared to the unosmicated specimens. Sequential treatments of osmicated specimens with tannic acid pH 7.0–9.0, and uranyl acetate, pH 4.1, enhanced the density of the elastin intensely, increased collagen staining moderately, but hardly increased the density of nuclei and microfibrils. In elastase-digested osmicated specimens, all tannic acid (pH 7.0)-uranyl acetate-reactive elastin was selectively removed. These results demonstrate that all the neutral and alkaline tannic acid-uranyl acetate methods can be used as a postembedment stain for elastin specimens fixed in glutaraldehyde and osmium tetroxide.     

Are young female gymnasts malnourished? An anthropometric, electrophysiological, and histological study

Competitive rhythmic sportive gymnastics have been accused of promoting an unphysiologic weight reduction which may progress to manifest anorexia nervosa. In this study, eight young female gymnasts who represented Norway in the European Championships in Rhythmic Sportive Gymnastics 1982 were examined for evidence of malnutrition. Ten girls, matched for age and height, served as controls. The examination included registration of anthropometric data (height, weight, and body-mass index), motor and sensory neurography and biopsies of the vastus lateralis muscle with exact measurements of muscle fibre areas on sections stained for myofibrillar ATPase activity. The mean body weight of the gymnasts did not differ from that of the control group or of a large series of age matched Norwegian females. This finding excludes the possibility of general malnutrition among the examined gymnasts. Muscle fibres of both types 1 and 2 were found to be smaller in the gymnasts than in the controls, with values of 3,404 microns2 vs 3,811 microns2 for type-1 fibres and 2,985 microns2 vs 3,942 microns2 for type-2 fibres respectively. Although contradictory to most previous reports, this finding suggests that the reduction in fibre size among the gymnasts might be an effect of physical training. There were some differences in neurographic parameters between the groups, but the mean values were all within normal ranges. The motor nerve conduction velocity in the proximal segments of the median and ulnar nerves was significantly slower in the gymnasts and, as a possible consequence of smaller muscle fibres, the motor responses were generally less in this group.     

Serotonin immunoreactivity in the intermediate lobe of the rat pituitary.

Immunocytochemical staining for serotonin (5-HT) in paraffin-embedded sections of rat pituitary resulted in the localization of reactive nerve fibres and cell bodies in the intermediate lobe. Immunostaining was also found in the anterior and posterior lobes. Labelled nerve fibres appear to enter the intermediate lobe from the neural lobe through the interlobular spaces. These fibres are relatively scarce and lightly stained. Neuroglandular contacts were identified between varicose nerve endings containing serotonin and immunoreactive perykarion. It is not clear whether intermediate lobe cells produced 5-HT themselves or, alternatively, these cells take in 5-HT from serotoninergic nerve terminals.     

Fluorescence microscopical visualization of elastic fibres using basic fuchsin

Summary We show that fluorescence microscopy after staining of tissue sections with basic fuchsin (BF) can be used successfully for the demonstration of elastic fibres. Using double staining with BF and antibodies reacting with microfibrils of elastic fibres (anti-SAP) we showed that BF reacts with the elastin core of elastic fibres and the elastin poor terminal branches of the subepidermal elastic fibre system. Small amounts of bound BF were easily seen by fluorescence microscopy (FL) but not by ordinary light microscopy. Both frozen sections and sections of paraffin embedded tissues could be stained. The BF-FL staining procedure is simple to perform and, due to its selectivity, it may be useful for detecting elastic fibres in various tissues at the light microscopical level.     

Fluorescence microscopical visualization of elastic fibres using basic fuchsin

We show that fluorescence microscopy after staining of tissue sections with basic fuchsin (BF) can be used successfully for the demonstration of elastic fibres. Using double staining with BF and antibodies reacting with microfibrils of elastic fibres (anti-SAP) we showed that BF reacts with the elastin core of elastic fibres and the elastin poor terminal branches of the subepidermal elastic fibre system. Small amounts of bound BF were easily seen by fluorescence microscopy (FL) but not by ordinary light microscopy. Both frozen sections and sections of paraffin embedded tissues could be stained. The BF-FL staining procedure is simple to perform and, due to its selectivity, it may be useful for detecting elastic fibres in various tissues at the light microscopical level.     

Coexistence of nitrergic and acetylcholinesterase (ACHE)-positive nerve structures of the phaesant spleen

The coexistence of neuronal NADPH-diaphorase and ACHE activities were investigated in the phaesant spleen by successive double histochemical staining of the same sections. Two types of nerve structures were found in pheasant the spleen: nerve cells and nerve fibres. NADPH-d and ACHE-positive nerve fibres in colocalization enter the spleen in its hilum in the vicinity of splenic artery branches and are gradually distributed in periarterial topography in the white pulp. Only NADPH-d positive nerve cells were seen around the splenic vessels. In the red pulp and splenic capsule, only ACHE-positive nerve fibres were present.     

Distribution of dopamine-beta-hydroxylase-like immunoreactivity in the rat pineal organ

The aim of the present investigation was to study the distribution in the rat pineal gland of dopamine-beta-hydroxylase (DBH) which is essential for the formation of the melatonin synthesis-regulating substance noradrenaline (NA). In 5- and 8-month-old male Sprague-Dawley rats DBH-like immunoreactivity (DBH-LI) was studied using polyclonal antibodies against DBH and the indirect immunofluorescent technique. DBH-LI was mainly located in pineal nerve fibres coming from the superior cervical ganglia. The intensity of the staining reaction was considerably lower than in non-pineal noradrenergic nerve fibres and the impression was gained by comparison of DBH-LI specimens with glyoxylic acid-treated sections that only approximately one third of the NA-containing intrapineal nerve fibres exhibited DBH-LI. There were no detectable differences in DBH-LI with regard to time of day and age of the animals. These results suggest that NA synthesis may be relatively low in intrapineal sympathetic nerve fibers and that the NA required for the regulation of pineal melatonin synthesis may, to a large degree, stem from the circulation. In addition to nerve fibres, some rare intrapineal cell bodies exhibited DBH-LI; in 5-month-old rats their numbers did not reveal significant differences between day and night. These cells do not appear to represent pinealocytes. They may be a special population of noradrenergic nerve cells perhaps belonging to an as yet unknown intrapineal regulatory system.     

莼菜(Brasenia schreberi)表皮腺毛细胞高尔基体中一种内含物的初步研究(简报)

在不经过任何特殊处理的常规生物样品中,高尔基体扁囊(Saccules)及囊泡(Vacuoles)中的内含物在电镜下常为低电子密度,而最近我们在莼菜(Brasenia schreberi)叶柄及叶片的表皮腺毛细胞中观察到带有高电子密度的高尔基体内含物。在扁囊中,这些内含物多呈波浪形(图版Ⅰ,图1)。这种特殊形态的高尔基体内含物以及这种未经任何特殊处理而显示出高尔基体中某些物质的现象是前人没有报道过的,本文就这种内含物的结构、性质以及其染色机制进行了初步探讨。     

CGRP-like immunoreactivity in sensory nerve endings of the Golgi tendon organ. A light- and electron-microscopic study in the grey short-tailed opossum (Monodelphis domestica)

Using light- and electron-microscopic immunohistochemistry, it was shown that primary sensory nerve endings in Golgi tendon organs of the grey short-tailed opossum (Monodelphis domestica) contain immunoreactivities to a polyclonal antibody directed against calcitonin gene-related peptide (CGRP). Myelinated afferent axons (6-9 microns in diameter) of the Golgi tendon organs stained moderately for CGRP. Sensory nerve endings within the sensory compartment of the Golgi tendon organs displayed electron-dense accumulations corresponding to dark-brown staining in adjacent semithin sections. On the outer surface of tendon organs C fibre bundles were observed showing CGRP-like immunoreactivity.     

Differential staining of nerve cells and fibres for sections of paraffin-embedded material in mammalian central nervous system

A differential staining method is described of myelinated fibres and nerve cell bodies applicable to sections of mammalian, including human, central nervous system specimens embedded in paraffin wax. Experimental and human necropsy material fixed in acetic paraformaldehyde in phosphate buffer was used. Sections of 15–20 m in thickness were obtained, attached to slides, deparaffinized and hydrated. After hydration, sections are oxidized (30 s) in 2% potassium permanganate, bleached (1 min) in 5% oxalic acid and rinsed in distilled water. Staining is for 2–5 h in the following solution: 0.06% thionin, 1% formaldehyde, 10% acetic acid in distilled water. Sections are subsequently washed in distilled water, dehydrated through 96% and absolute ethanol, cleared in eucalyptol and mounted in Eukitt. Using the method described in the present paper, a differential coloration of myelin and neurons is obtained. Myelinated fibres appear red, whereas nerve cell bodies and glial nuclei are stained blue. This procedure provides a high contrast between myelin and cells suitable for observation and photography of sections. Simultaneous and differential coloration of both myelin and cells is easily and directly obtained with constant and homogeneous results.     

The characteristic three-banded birefringence of ligamentum nuchae elastic fibres indicates ordered micellar texture

The ultrastructural organization of the elastic fibres of bovine ligamentum nuchae is still controversial (Kádár 1979; Sandberg 1976; Sandberg et al. 1981; Quintarelli et al. 1973; Gosline 1976). Polarization microscopy demonstrated convincingly, contrary to a recent negative report (Aaron and Gosline 1980), an ordered birefringent micellar texture (Schmidt 1939; Romhányi 1965; Fischer 1979). The three-banded anisotropic pattern of the fibres can be explained by the optical interference of two micellar textures of opposite orientation, one arranged circularly in the surface layer and the other oriented longitudinally in the axial core of the fibres (Romhányi 1965). Analysis of the underlying molecular mechanism for the anisotropic staining reaction of these fibres with Congo red led to the conclusion that this is due to a definite structural order of specific cross-linking segments within the random network of elastin. The anisotropic staining reaction of the elastic fibres with Congo red is described as the most simple method to demonstrate the three-banded birefringent pattern of ligamentum nuchae elastic fibres even in histologic sections.     

Protein gene product (PGP) 9.5 immunoreactivity in nerve fibres and pinealocytes of guinea-pig pineal gland: interrelationship with tyrosine-hydroxylase- and neuropeptide-Y-immunoreactive nerve fibres

This light-microscopic (LM) immunohistochemical study has evaluated the presence and distribution of the pan-neural and neuroendocrine marker protein gene product (PGP) 9.5 in pinealocytes and nerve fibres of guinea-pig pineal gland. The pattern of PGP 9.5-immunoreactive (ir) nerve fibres has been compared with that of fibres staining for tyrosine hydroxylase (TH) or neuropeptide Y (NPY). The vast majority of pinealocytes stained for PGP 9.5, although with variable intensity. PGP 9.5 immunoreactivity was localized in pinealocytic cell bodies and processes. Double-immunofluorescence revealed that PGP 9.5 immunoreactivity was absent from glial cells identified with a monoclonal antibody against glial fibrillary acidic protein (GFAP), PGP 9.5 immunoreactivity was also present in a large number of nerve fibres and varicosities distributed throughout the pineal gland. The number of TH-ir and NPY-ir nerve fibres was lower compared with those containing PGP 9.5 immunoreactivity. All fibres staining for NPY also stained for TH. NPY-ir nerve fibres were found to be much more numerous than previously reported for this species. The double-immunofluorescence analysis indicated that almost all TH-ir nerve fibres of the pineal gland contained PGP 9.5 immunoreactivity. However, few PGP 9.5-ir nerve fibres, located in the periphery and the central part of the gland, were TH-negative. A large number of PGP 9.5-ir fibres was concentrated in the pineal stalk. In contrast, TH-ir and NPY-ir nerve fibres were rare in this part of the pineal gland. Our data provide evidence that immunohistochemistry for PGP 9.5 may be a useful tool further to differentiate central and peripheral origins of pineal innervation. Furthermore, the staining of pinealocytes for PGP 9.5 may be exploited to study the three-dimensional morphology and the architecture of pinealocytes and their processes under various experimental conditions.     

Comparison of red and white muscles by cytophotometry of their muscle fibre populations

Synopsis Samples from two red muscles (vastus intermedius and vastus medialis) and two white muscles (biceps femoris and gluteus medius) were taken from four pigs. Serial transverse sections were reacted for ATPase and NADH oxidative activity. Sections were mapped with a projection microscope so that the staining intensity of individual fibres for the two reactions could be measured with a simple microscope photometer. Transmission values at 600 nm were converted to units of 0–10 for the range from darkest to lightest staining fibres on each section to cancel variation in staining intensity between sections. The aim of the study was to use simple cytophotometry instead of subjective judgement in the categorization of different histochemical types of muscle fibres. Cytophotometry enabled clear resolution of the major fibre types (types I and II using the ATPase reaction), partial resolution of more variable characteristics (NADH oxidative activity in type I and II fibres) and no resolution of subtle subtypes (IA and IB with the NADH oxidative reaction). However, between the major fibre types, cytophotometry revealed variable numbers of fibres with transitional characteristics. There were more of these fibres in red muscles. With sections reacted for ATPase, transmission values for low magnification fields containing 100 to 200 fibres were correlated (r=–0.91) with the ratio of type I:II fibres.     

Simultaneous demonstration of adrenergic and non-adrenergic nerve fibres

Summary A technique for simultaneous demonstration of adrenergic and non-adrenergic nerve fibres is described, using methylene blue staining and fluorescence microscopy after formaldehyde treatment. The procedure is applicable to whole mounts as well as to microtome sections.     

Visualization of Legionella pneumophila in paraffin sections using hexamine silver

A modification of Gomori's hexamine silver technique is given as a simple, reliable method for the nonspecific demonstration of Legionella pneumophila in paraffin sections. When tested against serogroups I to VI it was found that pretreatment with potassium dichromate rendered L. pneumophila demonstrable by the Gomori-Burtner hexamine silver solution when buffered to pH 7.8. Tissue was fixed in 10% buffered formalin and sections were cut at 3-5 microns. After treatment with 10% potassium dichromate for 1 hour at room temperature, sections are placed in the silver solution at 56 C until they develop a pale golden yellow color, at which point they are checked periodically under the microscope for optimal staining (approximately 3-4 hours). Sections are then toned, fixed and counterstained in 1% neutral red. The L. pneumophila coccobacilli stain black against a clear background, while nuclei stain red/black.