The interrelation of the size and substantivity of dyes; the role of van der Waals attractions and hydrophobic bonding in biological staining

Summary Using a pH signature criterion, it was found that whereas electrostatic attractions and repulsions were paramount in the binding of low molecular weight acid and basic dyes to tissue sections, high molecular weight dyes were also bound non-electrostatically.By studying the effects on staining of adding to aqueous dyebaths agents destroying the iceberg structure of water, the importance of hydrophobic bonding was established. It was noticed that the hydrophobic elastic fibres were stained by large dyes from dyebaths inhibitory both to electrostatic attractions and hydrophobic bonding (i.e. using acid dyes from alkaline aqueous-ethanol or aqueous-dimethylformamide dyebaths). This indicated that strong van der Waals attractions occurred, at least with some substrates. Supporting this idea was the observation that in tissue sections benzoylated before staining (i.e. made less acidophilic but more hydrophobic) additional structures were stained when using large acid dyes from alkaline aqueous-ethanol or aqueous-dimethylformamide dyebaths.Applications of the size-substantivity relationship were suggested, e.g. commenting on a standard stain for basic proteins; explaining the modes of action of traditional stains for elastic fibres and amyloid; rationalising the varied substantivities of tetrazolium salts; and finally suggesting guide lines for use in the design of new staining methods.     

In vivo imaging of the airway wall in asthma: fibered confocal fluorescence microscopy in relation to histology and lung function

Background

Airway remodelling is a feature of asthma including fragmentation of elastic fibres observed in the superficial elastin network of the airway wall. Fibered confocal fluorescence microscopy (FCFM) is a new and non-invasive imaging technique performed during bronchoscopy that may visualize elastic fibres, as shown by in vitro spectral analysis of elastin powder. We hypothesized that FCFM images capture in vivo elastic fibre patterns within the airway wall and that such patterns correspond with airway histology. We aimed to establish the concordance between the bronchial elastic fibre pattern in histology and FCFM. Second, we examined whether elastic fibre patterns in histology and FCFM were different between asthmatic subjects and healthy controls. Finally, the association between these patterns and lung function parameters was investigated.

Methods

In a cross-sectional study comprising 16 subjects (8 atopic asthmatic patients with controlled disease and 8 healthy controls) spirometry and bronchoscopy were performed, with recording of FCFM images followed by endobronchial biopsy at the airway main carina. Elastic fibre patterns in histological sections and FCFM images were scored semi-quantitatively. Agreement between histology and FCFM was analysed using linearly weighted kappa κ_w.

Results

The patterns observed in histological sections and FCFM images could be divided into 3 distinct groups. There was good agreement between elastic fibre patterns in histology and FCFM patterns (κ_w 0.744). The semi-quantitative pattern scores were not different between asthmatic patients and controls. Notably, there was a significant difference in post-bronchodilator FEV₁ %predicted between the different patterns by histology (p = 0.001) and FCFM (p = 0.048), regardless of asthma or atopy.

Conclusion

FCFM captures the elastic fibre pattern within the airway wall in humans in vivo. The association between post-bronchodilator FEV₁ %predicted and both histological and FCFM elastic fibre patterns points towards a structure-function relationship between extracellular matrix in the airway wall and lung function.

Trial registration

Netherlands Trial Register NTR1306     

Correlation of individual skeletal muscle fibres from “semithin” sections stained with p-phenylene-diamine and histochemical sections incubated for myofibrillar ATP-ase

Summary Muscle fibres in transverse semithin sections of osmium-fixed and plastic embedded muscle tissue stained with p-phenylene-diamine has been classified into three types (M, INT and H) and correlated to the same fibres in neighboring sequential preincubated myofibrillar ATP-ase sections. A close correlation, but no equality was found between the fibre types classified according to the ATP-ase and the p-phenylene-diamine methods. On the average, the content of mitochondria and fat droplets was highest in the ATP-ase classified type I fibres (dominated by M fibres), and higher in the endurance trained than in the untrained subjects.     

A comparison of tinctorial and immunohistological methods for the detection of fibrinoid change and fibrin deposition in the kidney

Synopsis Renal tissue from six cases with fibrinoid change or with glomerular fibrin deposits was examined by the standard tinctorial methods (Lendrum's Martius Scarlet Blue, phosphotungsticacid-Haematoxylin and Lendrum's picro-Mallory) for the demonstration of fibrin. It was found that all three methods should be used to detect its presence. Examination by immunofluorescence techniques indicated that little or no fibrinogen or fibrin need be present in areas giving positive tinctorial reactions.     

Nitric oxide synthase-immunoreactive axons innervating the guinea-pig lingual artery: an ultrastructural immunohistochemical study using elastic brightfield imaging

The ultrastructure of nitric oxide synthase-immunoreactive (NOS-IR) axons innervating the guineapig lingual artery was investigated by means of pre-embedding immunohistochemistry using an indirect peroxidase technique and diaminobenzidine. Sections ranging in thickness from 60 to 500 nm were ultrastructurally evaluated in elastic brightfield imaging mode. Thick sections (optimum at 300 nm) were advantageous for enhancement of the labelling intensity, whilst some subcellular details were better revealed by thin sections. NOS-IR axon terminals often contained aggregations of large, dense-cored vesicles, consistent with a previous light microscopical report on colocalization of NOS and vasoactive intestinal peptide-immunoreactivity in these fibres. NOS-IR axons formed direct neuro-muscular junctions (width less than 50 nm) at the outer surface of the tunica media, thus providing a structural basis for nitrergic vasodilation. In addition, NOS-IR axons made direct contacts with non-varicose and varicose segments of non-reactive axons, suggesting interneuronal communication between these elements.     

Transitional stages in the histochemical development of muscle fibres during post-natal growth

Synopsis Serial frozen sections of longissimus dorsi muscles from seven pigs at different live weights (13 to 127 kg) were reacted for ATPase by the calcium method at an alkaline pH and for NADH oxidative activity. One hundred muscle fibres from each animal were identified individually in serial sections and their staining intensity was measured with a microscope photometer at 600 nm. For each section, staining intensity of fibres (% tranmission) was measured and converted to the nearest one-tenth unit of the range from the darkest to the lightest staining fibres. Frequency of occurrence of fibre types was plotted on a 10×10 grid using the range co-ordinates for NADH oxidative activity (vertical) and ATPase activity (horizontal). The commonly recognized histochemical fibre types in this muscle appeared as crowded areas in the grid but, in many cases, these areas were part of a continuous L shaped distribution. In fibres having an ATPase staining intensity of 1.0 and 0.9 units of the range, a continuous but skewed distribution with regard to NADH oxidative activity was detected. In fibres with NADH oxidative activity of 0.6 to 1.0 units of the range, a continuous but irregular distribution with regard to ATPase activity was detected. Within this range, there was some evidence of a growth-related shift towards weaker ATPase activity.     

Myelogenesis and estimation of the number of axons in the anterior commissure of the chick (Gallus gallus)

Summary By use of light- and electron microscopy the anterior commissure of the chick was studied at different times during development. Between the 19th day of incubation and the 35th day after hatching the cross-sectional area of the anterior commissure, as determined from mid-sagittal sections, undergoes a 6-fold increase in size. Thereafter the area remains fairly constant. The total number of fibres in the anterior commissure was estimated to be 89000. The full complement of fibres is already present by the 19th day of incubation. Myelogenesis occurs mainly between the 19th day of incubation and the 35th day after hatching, concomitant with the increase in cross-sectional area. From the 35th day after hatching, myelinated fibres comprise approximately 40% of the total number of fibres. The median diameter of unmyelinated fibres is about 0.35–0.40 m. The median diameters of myelinated axons and fibres are 0.8–1.0 m and 1.1–1.3 m, respectively.     

Expression of an alpha cardiac-like myosin heavy chain in muscle spindle fibres

Summary In the present study we have investigated the reactivity of rat muscle to a specific monoclonal antibody directed against alpha cardiac myosin heavy chain. Serial cross sections of rat hindlimb muscles from the 17th day in utero to adulthood, and after neonatal denervation and de-efferentation, were studied by light microscope immunohistochemistry. Staining with anti- myosin heavy chain was restricted to intrafusal bag fibres in all specimens studied. Nuclear bag₂ fibres were moderately to strongly stained in the intracapsular portion and gradually lost their reactivity towards the ends, whereas nuclear bag₁ fibres were stained for a short distance in each pole. Nuclear bag₂ fibres displayed reactivity to anti- myosin heavy chain from the 21st day of gestation, whereas nuclear bag₁ fibres only acquired reactivity to anti- myosin heavy chain three days after birth. After neonatal de-efferentation, the reactivity of nuclear bag₂ fibres to anti- myosin heavy chain was decreased and limited to a shorter portion of the fibre, whereas nuclear bag₁ fibres were unreactive. We showed that a myosin heavy chain isoform hitherto unknown for skeletal muscle is specifically expressed in rat nuclear bag fibres. These findings add further complexity to the intricate pattern of isomyosin expression in intrafusal fibres. Furthermore, we show that motor innervation influences the expression of this isomyosin along the length of the fibres.     

Morphology and mechanics of the larynx of the pigeon (Columba livia L.): A drill-chuck system (Aves)

Summary The morphology and mechanics of the larynx and the ventral pharyngeal valves of the pigeon (Columba livia L.) are described. The glottal apparatus is an elastic ring of cartilage since the caudomedial ridges of the cricoid wings are tightly interconnected via the median cauda procricoidea. The bilateral arytaenoids can only hinge mediolaterad relative to the corpus procricoideus as a result of their particularly shaped articulation facets. The procricoid is tilted by changing the constriction of the ring. At rest, the glottis is held in an intermediate open position because the arytaenoids are kept along the dorsal ridges of the alae cricoideae in an elastic open rest position by the elasticity of the ring and of the lig. arytaenocricoideum. The large dilator muscle covers the apparatus as a continuous sheet. The constrictor muscle complex, however, has five discrete sections, each having a different origin. They all insert on the heavy interarytaenoid ligament and one of them is a sphincter. These five sections can constrict the cricoprocricoid ring, thus causing a forward tilting of the procricoid which forces the arytaenoids to hinge mediad. The glottal apparatus operates as a drill-chuck system in which the arytaenoids are taken as the graspers (chuck jaws). The model is studied by a movement- and electromyographic analysis of three common glottal actions: respiration, reaction to water, and reaction to touch. The elastic open rest position of the glottis is found in anaesthetized birds. The drill-chuck model is tested by the comparison of the recorded myogram pattern with the myogram pattern that must follow from the model for the moving structures. The ventral pharyngeal valves are erected by the long submucosal m. hyovalvularis, running between the basihyal and the dorsal side of the gland. cricoarytaenoidea in the valves. The valves are pulled down to the floor of the pharynx by a network of collagen and elastic fibres connected to the small m. tracheovalvularis, which runs down to a medium strand attached to the first tracheal ring. These two subsystems of the floor of the pharynx operate independently during respiration. But, in case of pecking and drinking they show combined actions which place them as parts of a highly integrated system.This paper is number IV of a series on Morphology and mechanics of the feeding system of the pigeon (Columba livia L.)     

Ultrastructure of free nerve endings in respiratory and squamous epithelium on the rat nasal septum

The distribution of nerve fibres in the mucosa of the nasal septum of the rat was investigated by means of transmission electron microscopy on transverse and tangential ultrathin sections. Near the basement membrane of respiratory and squamous epithelium, a rather dense network of unmyelinated nerve fibres occurs. Some fibres in the respiratory epithelium ascend between the epithelial cells to reach up to the tight junctions. These fibres appeared in transverse sections to end as hooks or boutons, sometimes with branches. These shapes resemble the free nerve endings that are considered to act as nociceptors. The small intraepithelial fibres, with diameters of about 0.5–1 m, contain both dense granules and clear vesicles comparable to synaptic vesicles. Substance P was found in dense granules in basal fibres; vasoactive intestinal peptide was absent throughout the epithelium. Acetylcholinesterase activity was observed closely associated with the basal fibres; the apical fibres showed little if any activity. Membrane specializations pointing to an efferent function as well as structures usually associated with mechanoreceptive functions were lacking in both respiratory and squamous epithelium.Part of this work was presented at the Annual Conference of the Netherlands Society for Electron Microscopy, 28–29 Nov 1985, Wageningen, The Netherlands. See Spit BJ and Hendriksen EGJ (1986) Ultramicroscopy 19:102–103     

Ultrastructure of arterioles in the cat brain

Summary A total of 110 arterioles were examined in the brains of cats; different sites were studied including the cortex, putamen, pons and crus cerebri. No internal elastic laminae were seen in the subendothelial space, although occasional fragments of elastic material were present in the larger arterioles. The media was composed of one, two or three layers of smooth muscle cells which interlocked in such a way that the vessel wall thickness was constant. Numerous tight junctions were seen between adjacent smooth muscle cells and between the endothelium and smooth muscle cells. Apart from the usual cell organelles, the smooth muscle cells of arterioles had numerous dense patches on the cell surface. The structure of the adventitia varied according to the diameter of the vessel and the site in the brain; it contained adventitial cells, bundles of collagen fibres and nerve fibres. Innervation of arterioles was more constant in the brain stem than in the cortex. Metarterioles had less specialised, atypical smooth muscle cells, a discontinuous media and numerous, extensive myoendothelial tight junctions; they were not innervated by nerve fibres. The diameter of metarterioles was less than 10 m whereas that of arterioles was 10–45 m. The possible functional aspects of arteriolar innervation are discussed.     

Conditions for the immunohistochemical demonstration of complement factor C3 in formaldehyde-fixed and paraffin-embedded renal tissues

Summary Paraffin sections of formaldehyde-fixed renal biopsies were labeled for complement C3 by a polyclonal rabbit antibody to human complement C3, by the peroxidase-antiperoxidase complex (PAP) and the avidin-biotin peroxidase complex (ABC) techniques, respectively. All tissues had C3 deposits according to direct immunofluorescence on fresh frozen sections. Staining for muramidase was introduced as an intrinsic control for the degree of tissue proteolysis after the necessary trypsin digestion prior to the immunoenzyme labeling. The results indicated that even minute deposits of C3 could be detected in paraffin sections by the ABC method, which was more sensitive than the PAP technique; the ABC method allowed a maximal dilution of 12,400 of the primary antibody as compared to 1800 for the PAP technique.     

Lack of ''acid reversal'' of myofibrillar adenosine triphosphatase in masticatory muscle fibres of rhesus monkeys

Summary Myofibrillar adenosine triphosphatase (ATPase) activity was demonstrated in sections of masseter and temporalis muscles and of selected limb muscles of adult rhesus monkeys. Incubations were performed either with no pre-treatment or after prior incubation in alkaline media (pH 10.2–10.4) or acidic media (pH 3.8–4.6). Without pre-treatment, fibres having high or low ATPase activity were observed in limb and masticatory muscles. Following alkaline pre-incubation the difference between high and low ATPase of limb muscle fibres is accentuated, whereas pre-incubation in acidic media (pH 4.3) results in inhibition of high and potentiation of low ATPase activities (acid reversal). While pre-incubation of masticatory muscle sections at pH 10.2 accentuates differences in ATPase activity, pre-incubation at pH 10.4 abolishes ATPase activity. In contrast, masticatory muscle fibres showed no reversal of ATPase activity following acidic pre-incubation (pH 4.3). Pre-incubation at pH 3.8 abolished the ATPase activity of both limb and masticatory muscle fibres. The biochemical basis for the differences in ATPase histochemistry between masticatory and limb muscles is not known.     

Further histochemical properties of rabbit skeletal muscle fibres

Summary The histochemical activities of succinic dehydrogenase (SDH), creatine kinase (CK), sarcoplasmic reticular ATPase (SR-ATPase) and myosin ATPase were studied in serial sections of rabbit adductor muscle. Three fibre types were distinguished depending upon the distribution of the enzyme activities. The type II white fibres posessing minimal SDH showed high myosin ATPase, SR-ATPase and ATPase dependent CK activities. Red oxidative fibres showing high SDH fell into two distinct groups: One category had mainly a peripheral localization of SDH and showed an enzymatic profile identical to that of type II white fibres. The second category of red fibres displayed both a homogeneous distribution of small diformazan granules throughout the fibre as well as a sub-sarcolemmal collection when tested for SDH activity but possessed very low amounts of reaction product of the various enzymes of the energetic metabolism studied. Since it is well established that the myosin ATPase of a fibre correlates with its contraction time, the present histochemical investigation provides further support for this concept by demonstrating the presence of high SR-ATPase and ATPase dependent CK activities in all white and red fibres rich in myosin ATPase.     

Cross bridge slippage induced by the ATP analogue AMP-PNP and stretch in glycerol-extracted fibrillar muscle fibres

Glycerol-extracted insect fibrillar muscle fibres in rigor exhibited both an elastic and a plastic phase in the length-tension diagram. The transition between these phases took place at a critical tension, the yield point or elastic limit. In the plastic phase the apparent static elastic modulus became zero, whereas the immediate elastic modulus (measured by rapid length changes completed within 4 ms) exhibited no abrupt change at the yield point. The tension value of the yield point (but not immediate stiffness) was lowered by addition of AMP-PNP and was partially restored by washing out AMP-PNP. The dependence of the critical tension at which plastic flow begins on cooperative cross bridge behaviour is discussed in terms of breaking and reforming acto-myosin linkages. Evidence is presented that addition of AMP-PNP induces slippage of cross bridges on the actin filament by affecting the interaction between myosin and actin.     

Electron probe X-ray microanalysis of human muscle biopsies

Summary The elemental composition of human muscle fibres have been determined by electron probe microanalysis. In order to distinguish between different types of fibres, two approaches were used. In one approach individual fibres were isolated, portions of them used for a typing by histochemical methods and the main part used for X-ray microanalysis. In the other approach the muscle biopsy was serial-sectioned, some sections used for a histochemical typing and the others (16 m thick cryosections) used for X-ray microanalysis in the electron microscope.The comparison of the ratios between P, S and K in Study No. 1 and 2 indicates different concentrations of sulphur in the subsarcolemmal zone and in the interior of the fibre. Both routes give information on all elements (except the ten lightest ones) contained in the fibres or in sections of them, provided the concentration is high enough. In order to obtain quantitative data, expressed as mmol/kg_dw, the spectra of the specimens were compared to those of standards of known composition and the data subjected to a so called ZAF-correction (corrections for the atomic number effect, absorption of X-rays in the specimen and secondary fluorescence). Quantitative data concerning phosphorus, sulphur, chlorine and potassium were obtained in Study No. 2. A significantly higher sulphur concentration was found in type IIA muscle fibres as compared to those of type I.     

Three-dimensional architecture of chromosome fibres in the crane fly: co-oriented autosomal bivalents and amphitelic sex univalents during prometaphase

A technique for the fixation of cells during live observation (Nicklas et al. 1979) was used to investigate chromosomes which were moving at the time of fixation. Chromosome fibres were reconstructed by tracking their microtubules in longitudinal serial sections. A considerable proportion of non-kinetochoric microtubules (free microtubules, fMTs) is skewed with respect to the fibre axis. These skew fMTs contribute to the degree of disorder. It was found that the difference in the relative proportion of skew fMTs between active fibres (oriented in the direction of movement) and passive fibres (oriented backwards) is significantly correlated with the chromosome velocity (correlation coefficient r=0.796, P=0.01). It can be concluded that the pulling force generated in the chromosome fibre is a function of skew fMTs.     

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.     

Glutamate-immunoreactive climbing fibres in the cerebellar cortex of the rat

The climbing fibre system, one of the two main excitatory inputs to the cerebellar cortex, is anatomically and physiologically well characterized, while the nature of its neurotransmitter is still a matter of debate. We wished to determine whether glutamate-immunoreactive profiles with the morphological characteristics of climbing fibres could be found in the rat cerebellar cortex. For this purpose, a monoclonal anti-glutamate antibody has been used in combination with a sensitive postembedding immunoperoxidase method on semithin sections or in combination with a postembedding immunogold method on ultrathin sections. At the light microscopic level, climbing fibres appeared as strongly stained fibrous profiles, chains of interconnected varicosities or heavily labelled dots of various sizes, often in close apposition to principal Purkinje cell dendrites. At the electron microscopic level, certain labelled varicosities or more elongated profiles resembling climbing fibre terminals were in synaptic contact with dendritic spines of Purkinje cells. Quantitative analysis of gold particle densities showed that such elements were about three to four times more heavily labelled than their postsynaptic partners. The results obtained in this study demonstrate that at least a subset of climbing fibres and their terminals contain relatively high levels of glutamate-like immunoreactivity and provide additional evidence for a role of glutamate as transmitter in these cerebellar afferents.