Correcting the axial shrinkage of skeletal muscle thick sections visualized by confocal microscopy

Confocal microscopy is a suitable method for measurements and visualization of skeletal muscle fibres and the neighbouring capillaries. When using 3D images of thick sections the tissue deformation effects should be avoided. We studied the deformation in thick sections of the rat skeletal muscle from complete stacks of images captured with confocal microscope. We measured the apparent thickness of the stacks and compared it to the slice thickness deduced from calibrated microtome settings. The ratio of both values yielded the axial scaling factor for every image stack. Careful sample preparation and treatment of the tissue cryosections with cold Ringer solution minimize the tissue deformation. We conclude that rescaling by the inverse of the axial scaling factor of the stack of optical slices in the direction of the microscope optical axis satisfactorily corrects the axial deformation of skeletal muscle samples.     

A simple procedure for demonstrating the overall morphology of fibroblasts in routine histological preparations of adult tissues,using silver impregnation

A silver impregnation procedure for the total visualization of fibroblasts, with all their processes, is described. The method is applicable to routine formalin-fixed paraffin sections, or to formalin-fixed or native cryostat sections. The results are illustrated with examples from loose and dense connective tissue. Fibroblasts are visualized as cells with long, branching dendritic processes. The fibroblasts maintain contact with each other via these processes, thus forming a reticulum of cells, even in adult connective tissue.     

Neuronal imaging with colloidal gold

Colloidal gold is easily prepared, and readily adsorbs to a number of immunoreagents and other proteins for a wide variety of uses for neuronal visualization. Gold probes serve a role as immunolabels for both light and electron microscopy. As an ultrastructural immunocytochemical marker for detection of proteins, peptides or amino acids, gold can be used for immunostaining thick or thin sections prior to embedding, or for immunostaining ultrathin sections after embedding tissue in conventional or unusual embedding matrices. By virtue of its particulate nature, gold as an immunolabel facilitates a semi-quantitative analysis of relative antigen densities on ultrathin sections. Various combinations of different size gold particles or dual immunolabelling with enzymatic immunolabels together with colloidal gold or silver-intensified gold serve well for ultrastructural immunocytochemical localization of two antigens in the same tissue section. Colloidal gold can be detected with light microscopy, transmission and scanning electron microscopy, and with confocal laser microscopy. Silver intensification allows detection of gold at both the light and electron microscope level, and increases the sensitivity of immunogold procedures. Colloidal gold is useful as a tracer for physiological studies of transport and internalization in neurons in vivo and in vitro; computer-assisted video imaging techniques allow detection and tracking of single gold particles in living cells.     

Lectins bind differentially to cilia and microvilli of major and minor cell populations in olfactory and nasal respiratory epithelia.

Binding of colloidal gold-conjugated lectins was studied in cilia and microvilli of rat olfactory and respiratory epithelia. This was done in sections of rapidly frozen, freeze-substituted specimens embedded in Lowicryl K11M or, for wheat germ agglutinin (WGA) alone, in deep-etched replicas. Olfactory dendritic endings and cilia labeled with WGA and faintly with soybean agglutinin (SBA); olfactory supporting cell microvilli bound only Dolichos biflorus agglutinin (DBA). Microvilli of an infrequent cell bound peanut agglutinin (PNA), SBA, and WGA. These microvilli labeled more strongly with the last two lectins than the olfactory cilia. Respiratory cilia bound WGA and, somewhat more weakly, PNA; microvilli of ciliated respiratory cells bound all four lectins. Visualization of specific labeling improved after preincubation of sections with neuraminidase, except for DBA where lectin binding was abolished. PNA labeling was seen only after neuraminidase preincubation. The densities of membrane surface particles that labeled with WGA corresponded with those of fracture plane particles in a quantitative freeze-fracture, deep-etch analysis. Therefore, a considerable fraction of the WGA-bound particles could reflect transmembrane proteins in olfactory dendritic endings and cilia and in respiratory cilia. The possible nature of these particles is discussed.     

Post-embedding cytochemistry with gold-labelled reagents: a review

The detection of antigens and glycoconjugates with the protein A–gold and the lectin–gold techniques, respectively, is reviewed. Special attention is directed to the necessary conditions for fixation and embedding as well as to the staining procedures of tissue sections for light and electron microscopy.     

Immunogold labeling of insulin growth factor-I receptors in elderly human skeletal muscle

Age-associated muscle wasting, or sarcopenia, can be delayed or reversed with interventions, including exercise and pharmaceutical agents. Mapping morphometric changes in the skeletal muscle insulin growth factor 1 receptor can provide valuable information regarding mechanisms controlling muscle protein metabolism. Immunocolloidal gold labeling is a powerful immunocytochemistry procedure for detecting antigens at the ultrastructural level, providing plausible biological markers of cell and tissue adaptations to stimuli. The intent here was to employ immunogold labeling to identify, localize, and quantify the insulin growth factor receptor-I (IGF-IR) in elderly human skeletal muscle. Needle biopsy specimens of the leg vastus lateralis muscle were fixed with 1% glutaraldehyde and 4% paraformaldehyde, dehydrated, and embedded in LR white resin. Pilot experiments were carried out to establish optimal dilutions of primary and secondary antibodies and to employ controls to establish staining specificity. The 6 nm gold particles were first evident when viewed at transmission electron microscopy (TEM) magnifications at 54,000x and clearest at 71,000x. Consistencies were noted in the staining patterns, with the majority of particles lying in proximity to the myofilaments. Gold particles were also found randomly along the outer membrane of the sarcolemma and the mitochondrial membranes. National Institutes of Health (NIH) Image 1.55 version software was used to measure receptor density (NIH, Bethesda, Md., USA). It appears that immunogold labeling of postembedded tissue samples is a sensitive method for detecting IGF-I receptors at the ultrastructural level.     

Embedding of large specimens in glycol methacrylate: prerequisites for multi-signal detection and high-resolution imaging

Acrylic resin mixtures are commonly used to study microscopic sections of biological specimens, giving the advantage of good morphological preservation. Existing embedding protocols, however, are suitable for tissue blocks, not exceeding 1 mm in thickness. We have developed a protocol to embed larger specimens (up to 2 cm(3)) in Technovit 8100. This medium allowed us to perform classic histological (trichrome), silver, as well as immunohistochemical staining, needed for multi-signal detection at high-resolution imaging to reconstruct a three-dimensional interpretation of a serially sectioned muscle. The technique was applied to reconstruct the semitendinosus muscle of a fetal pig, 44 days post conception, featuring connective tissue, intramuscular nerves, blood vessels, and muscle fibre types. For the reconstruction, a technique was used that enabled us to insert high-resolution images of histological details into low-resolution images of the entire muscle.     

Fluorescent imaging of endothelial glycocalyx layer with wheat germ agglutinin using intravital microscopy

Endothelial glycocalyx (GCX) is located on the apical surface of vascular endothelial cells and is composed of a negatively‐charged network of proteoglycans and glycoproteins. The GCX plays an important role in maintaining the integrity of vascular walls and preventing leakage of plasma. Therefore, degradation of the GCX is believed to lead to pathological leakage of plasma. Because the GCX is a very thin layer, its ultrastructural image has been demonstrated on electron microscope. To explore the function of the GCX, it should be visualized by a microscope in vivo. Thus, we developed in vivo visualization technique of the GCX under fluorescence microscopy using a mouse dorsal skinfold chamber (DSC) model. To label and visualize the GCX, we used fluorescein isothiocyanate (FITC)‐labeled lectin, which has a high specificity for sugar moieties. We examined the affinity of the different lectins to epivascular regions under an intravital fluorescent microscope. Among seven different lectins we examined, FITC labeled Triticum vulgaris (wheat germ) agglutinin (WGA) delineated the GCX most clearly. Binding of WGA to the GCX was inhibited by chitin hydrolysate, which contained WGA‐binding polysaccharide chains. Furthermore, the septic condition attenuated this structure, suggesting structural degradation of endothelial GCX layer. In conclusion, FITC‐labeled WGA lectin enabled visualization of endothelial GCX under in vivo fluorescence microscopy. Microsc. Res. Tech. 79:31–37, 2016. © 2015 Wiley Periodicals, Inc.     

Increasing membrane contrast by means of imidazole-osmium post-fixation as exemplified by skeletal muscle fiber

Until now, the interpretation of findings derived from investigations on membrane structures (T tubules, sarcoplasmic reticulum, the Golgi apparatus) in thick sections of mammalian muscle tissue has been limited in TEM due to the lack of sharp resolution of the membrane contours. This article shows how the imidazol-osmium post-fixation of tissue blocks can be used to achieve well-contrasted, sharply defined membrane contours. Therefore, unstained sections from imidazol-osmium post-fixed tissue can be examined immediately. But protein structures (e.g., ribosomes) remain uncontrasted with this technique. If needed, it is possible to visualize the protein structures by conventional section staining with uranyl acetate and lead citrate. This method is suitable for both ultrathin and thick sections (>150 nm).     

Three-dimensional analysis of neuronal geometry using HVEM

There is a need for an electron microscopic method for visualization of selectively stained neurons and neuronal processes with higher resolution than can be obtained with the light microscope, but using thick sections that allow visualization of the three-dimensional structure of the neuron. Such a method is required for measurement of the geometry of neurons, and this information is needed to test theoretical predictions on the way in which electrical signals of synaptic origin are processed by the cells. The high voltage electron microscope (HVEM) is well suited to this application, because of its high resolution and ability to form images of thick sections. Use of this instrument requires development of selective stains that can produce diffuse cytoplasmic staining of specific cells or cell populations on the basis of their functional properties. Several such methods currently being employed for light microscopic work can be used directly in the high voltage electron microscope or can be made useful by relatively minor alterations. These include intracellular staining with horseradish peroxidase, axonal tracing with Phaseolus vulgaris leukoagglutinin (PHA-L), and immunocytochemical staining for specific cell markers known to stain the cytoplasm of certain cell populations. Cells stained intracellularly by microinjection of horseradish peroxidase during physiological recording experiments may be stained in thick (ca. 50 μm) sections cut on a vibratome or similar instrument and stained in the standard way, using methods designed for light microscopy. The sections are then postfixed in osmium tetroxide and embedded in epoxy plastic. Sections cut from these blocks at thicknesses of from 1 to 5 μm using a dry glass knife may be examined directly in the HVEM with no further staining. This produces a very clear image of the cell on a relatively unstained background. This method provides more than adequate resolution of the boundary of the neuron, allowing measurement of neuronal processes to better than 10-nm precision. Similar results are obtained when the same method is applied to axonal tracing using PHA-L. In this case, the exogenously applied marker is used to label a small population of nearby neurons and to trace their connections with other cells at a distance. The lectin is detected by immunocytochemistry, but the selective contrast of the image is adjustable because the concentration of antigen in the cell is largely controlled by the experimenter. The lectin is distributed diffusely in the cytoplasm in a pattern identical to that of intracellular staining, so like intracellular staining, it reveals the overall shape of the cell. Immunocytochemical labelling using endogenous antigens known to be distributed in the cytoplasm of specific neurons produced inadequate control of selective contrast when prepared in this manner. Instead, 1–10μm sections cut from blocks of nervous tissue were embedded in polyethylene glycol, stained using a combedded in polyethylene glycol, stained using a combination of immunocytochemistry and histochemical intensification methods, and embedded in plastic on the grid. This method, which is also suited for staining with poorly penetrating markers such as colloidal gold, may also prove useful in a variety of other situations requiring the intensification of selective contrast.     

Preparing sections of skeletal muscle for transmission electron analytical microscopy (TEAM) of diffusible elements.

Comparative morphological examination and elemental analysis were carried out in structural compartments of sections of skeletal muscles. These had been prepared either by conventional plastic embedding technique or by various methods of cryo-ultramicrotomy. The analyses were performed in a Philips EM 301 with an Edax energy-dispersive X-ray spectrometer. Spectra obtained from sections of plastic-embedded muscle depended on the reagents used for fixation and staining and were absent if these were omitted. Brief fixation with glutaraldehyde resulted in gross ionic changes, and sectioning of frozen material with trough liquid led to extraction of elements. Sections cut from unfixed and frozen muscle without trough liquid showed numerous peaks. (Mg, P, S, Cl, K, Ca). In the superficial parts of the fibres of freeze-dried sections reproducible spectral differences were found between different structures. Thus, rapid freezing of unfixed tissue, dry cutting in the frozen state, and freeze-drying should be the procedure of choice if data on diffusible ions are desired.     

Silver staining of collagen and reticulin fibres and crebral capillaries by means of physical development

A method for demonstrating collagen and reticulin fibres and the network of brain capillaries has been worked out on the basis of a new silver staining principle. It may be applied to sections as thick as 200 μm from both frozen and embedded material. The principle is as follows: the 1,2-glycol groups of tissue elements, including connective tissues, are converted to aldehyde groups by periodic acid. The aldehyde groups form colloidal silver particles in an ammoniated silver nitrate solution of pH 13. At this pH value, however, it is only the connective tissue that can bind the colloidal silver which is being formed. After the silver ions have been washed out of the sections, the silver grains deposited in the connective tissue are finally enlarged to microscopic dimensions by physical development.     

A systematic approach to the discovery of new proteins of ultrastructural interest

A systematic approach to the discovery of new proteins of ultrastructural interest is discussed. It involves the merging of monoclonal antibody technology with immunocytochemical technology, particularly immunoelectron microscopy. In this approach, monoclonal antibodies are raised to a cellular preparation that can be grossly heterogeneous in its protein composition. The hybridoma culture fluids are screened by immunocytochemistry for the ultrastructural localization of their antibodies. Those monoclonal antibodies that show specific ultrastructural localizations of interest are then selected for further investigation. The antigen to which a given monoclonal antibody is directed is then identified by immunoprecipitation and immunoblotting with that antibody. By this approach, two new striated muscle proteins of ultrastructural interest have been discovered and are named zeugmatin and enactin. The former is a protein of over 500 kD localized by immunoelectron microscopy to the Z-bands, the latter of 245 kD localized to the N₁ line of striated muscle.     

Immunohistochemical myofiber typing and high-resolution myofibrillar lesion detection in LR white embedded muscle

We have developed a method of fixing, embedding, sectioning, and staining that allows high-resolution detection of myofibrillar structure and myosin immunocytochemical muscle fiber typing in serial semithin sections of LR White plastic embedded muscle at the light microscopic level. Traditional approaches, such as cryostat sections, permit fiber typing, but small myofibrillar lesions (1-3 sarcomeres) are difficult to detect because of section thickness. Semithin sections of hydrophobic resins do not stain well either histochemically or immunocytochemically. Electron microscopy can resolve lesions and discriminate fiber types based on morphology, but the sampling area is small. Our goal was to develop a rapid method for defining both fiber type and high-resolution primary myofibrillar lesion damage. Mild fixation (1-4% paraformaldehyde, 0. 05-0.1% glutaraldehyde) and embedment in a hydrophilic resin (LR White) were used. Myofibrillar structure was extremely well preserved at the light microscopic (LM) level, and lesions could be readily resolved in Toluidine blue stained 500-nm sections. Fiber type was defined by LM immunomyosin staining of serial plastic semithin sections, which demonstrated reciprocal staining patterns for "fast (Sigma M4276) and "total" (skeletal muscle) myosins (Sigma M7523).     

Investigation of staining, polarization and fluorescence-microscopic properties of myoendothelial cells

Electron microscopists have described in vascular endothelial cells filaments which resemble myofibrils. These fibrils cannot be demonstrated with conventional staining techniques. On the basis of previous investigations of relations between dye structure and affinity for muscle fibres, a staining method for demonstration of myoendothelial cells by direct, polarization and fluorescence microscopy has been developed. Carnoy-fixed paraffin sections were treated consecutively with Kernechtrot, tannic and phosphomolybdic acid and counter-stained with Levanol fast cyanine 5RN. This procedure stained myoendothelial cells and muscle fibres deep blue, connective tissues yellow and nuclei pink. For polarization and fluorescence-microscopic studies thiazine red R was substituted for Levanol fast cyanine 5RN and Kernechtrot was replaced by Mayer's acid hæmalum. The light-microscopic characteristics of fibrils in endothelial cells and in smooth muscle cells were identical. Correlation of light-microscopic observations with electron-microscopic data supported the conclusion that the staining procedure indeed demonstrates myofilaments in endothelial cells. Formation of multiple layers of myoendothelial cells was observed in areas of thickening of the intima, while the cells in the deeper layers became indistinguishable from smooth muscle cells. These findings support earlier concepts of the transformation of endothelial cells into intimal muscle cells.     

Expression of Leu-19 (CD56, N-CAM) and nitric oxide synthase (NOS) I in denervated and reinnervated human skeletal muscle.

Neural cell adhesion molecule (N-CAM, Leu-19, CD 56) expression appears during muscle fiber regeneration and after denervation. Sarcolemma-associated nitric oxide synthase (NOS) I, however, disappears from denervated myofibers. The dynamics of expression of both proteins were studied in 5 cases of acute/subacute denervation, 28 cases of chronic denervation with and without collateral reinnervation, 5 cases of the intermediate type spinal muscular atrophy (SMA 2), and in 2 normal biopsies. NOS I and its NADPH diaphorase (NADPHd) activity disappeared from the sarcolemma region shortly after denervation, and before the appearance of denervation atrophy. N-CAM was found diffusely distributed in the sarcoplasm at the most severe phase of denervation atrophy in the majority of highly atrophic fibers. During reinnervation, NOS I expression remained absent and in part of the cases the target/targetoid phenomenon appeared. In parallel with the increase in volume of the reinnervated muscle fibers, the intensity of N-CAM immunoreactivity decreased progressively. After full restitution of muscle fiber caliber, the target/targetoid phenomenon and N-CAM immunostaining disappeared completely, and, finally, NOS I reappeared in the sarcolemma region. The sarcolemmal expression of dystrophin and dystrophin-associated proteins was unchanged during denervation. NOS I was completely absent in children with SMA 2, since the protein does not appear before 5 years of age in skeletal muscle, while N-CAM was very intensely expressed in the sarcoplasm of highly atrophic denervated muscle fibers. In conclusion, this study suggests that innervation is an important factor for selective gene expression and positioning of NOS I and N-CAM in skeletal muscle and gives practical information for the assessment of the phase and developmental stage of the denervation and reinnervation process.     

Myotonic dystrophy protein kinase of the cardiac muscle: evaluation using an immunochemical approach

Myotonic dystrophy (DM) is an inherited multisystem disorder characterized by the presence of a high polymorphic expansion of trinucleotide (CTG) repeat in the 3' untranslated region of the DM protein kinase (DMPK) gene. However, the role of myotonic dystrophy protein kinase (DMPK) has yet to be elucidated. Studies aimed to discover possible physiological targets of DMPK indicated several subcellular localization sites, such as neuromuscular junctions, myotendinous junctions, and terminal cisternae of the sarcoplasmic reticulum in the skeletal muscle and intercalated discs in the cardiac muscle. Here, we extend our previous observations on the localization of DMPK at gap junction (GJ) level in the heart, taking advantage of the polyclonal peptide-specific anti-DMPK antibodies raised against two different domains of the protein. DMPK was detected by immunofluorescence at the intercalated disc level by both antibodies. Double immunofluorescence staining experiments performed with each anti-DMPK and anti-connexin43 showed colocalization of the two antigens. Immunoblot analysis of partially purified GJs showed co-sedimentation of DMPK and connexin43. We conclude that GJs are a genuine localization site of DMPK. Given the known regulation exerted by protein kinases on assembly, trafficking, gating, and disassembly of connexins, such a localization may be relevant to the functional role of connexins. DM is the most common muscular dystrophy in adults, and is known by the cardiac involvement that is a common feature in DM patients. Localization of DMPK at GJ in relation to DM is also briefly discussed.     

Leptin‐like immunoreactivity in the muscle of juvenile sea bass (Dicentrarchus labrax)

The mammalian hormone, leptin, is mainly synthesized in adipose tissue along with other tissues. Leptin plays a role in numerous processes such as in the control of food intake, homeostasis, immune function and reproduction. In this study, we detected and localized leptin immunoreactivity to the muscle of early juvenile sea bass (Dicentrarchus labrax) by Western blot analysis and immunohistochemistry. A leptin immunopositive band with a molecular weight of ～16 kDa, corresponding to mammalian leptin, was identified in trunk skeletal muscle homogenate. Furthermore, leptin immunopositive cells were detected in the endomysium of skeletal muscular fibers. These cells showed immunostained cytoplasmic granules and roundish and oval nuclei. The most intense immunostaining was observed in the endomysial space among the superficial red muscular fibers of the trunk. These findings suggest that in early juvenile sea bass, leptin is mostly produced by skeletal muscles. Therefore, during the developmental stage lacking adipose tissue, skeletal muscles can be considered an important source of leptin. As already suggested in mammals, we can hypothesize the potential roles of leptin not only in energy expenditure for muscle contraction but also during muscle differentiation and growth. Microsc. Res. Tech. 73:797–802, 2010. © 2010 Wiley‐Liss, Inc.     

A simple technique of image analysis for specific nuclear immunolocalization of proteins

Colocalization of fluorescent signals in confocal microscopy is usually evaluated by inspecting merged images from different colour channels or by using commercially available software packages. We describe in this paper a simple method for assessment of nuclear localization of proteins in tissue sections through confocal immunolocalization, propidium iodide counterstaining and image analysis. Through a macro command developed for the public domain, Java‐based software imagej , red, green, blue (RGB) images are automatically split in the red and green channels and a new image composed of the nonblack pixels coincident in both channels is created and inverted for better visualization. This method renders images devoid of both, extranuclear staining and background, thus emphasizing the nuclear signal. The resulting images can easily be used for comparison or quantification of the results. Given the simplicity of the technique and the worldwide diffusion of the software utilized, we think that this method could be useful in order to define standards of colocalization in confocal microscopy.