Staining of Muscle Tissue for Quantitative Studies and Automated Morphometry

Seventy-six staining tests were carried out on paraffin sections of human and animal muscle to find a suitable staining method for quantitative morphometry of muscle fibers. The results were evaluated under the light microscope, on black and white photomicrographs and on an image analysing computer, the Quantimet 720. A brilliant scarlet-phosphotungstic acid-tartrazine method is described and recommended for automated morphometry after additional testing on 140 sections of developing human muscle using the Quantimet 720.     

Three-dimensional temporomandibular joint muscle attachment morphometry and its impacts on musculoskeletal modeling

In musculoskeletal models of the human temporomandibular joint (TMJ), muscles are typically represented by force vectors that connect approximate muscle origin and insertion centroids (centroid-to-centroid force vectors). This simplification assumes equivalent moment arms and muscle lengths for all fibers within a muscle even with complex geometry and may result in inaccurate estimations of muscle force and joint loading. The objectives of this study were to quantify the three-dimensional (3D) human TMJ muscle attachment morphometry and examine its impact on TMJ mechanics. 3D muscle attachment surfaces of temporalis, masseter, lateral pterygoid, and medial pterygoid muscles of human cadaveric heads were generated by co-registering measured attachment boundaries with underlying skull models created from cone-beam computerized tomography (CBCT) images. A bounding box technique was used to quantify 3D muscle attachment size, shape, location, and orientation. Musculoskeletal models of the mandible were then developed and validated to assess the impact of 3D muscle attachment morphometry on joint loading during jaw maximal open-close. The 3D morphometry revealed that muscle lengths and moment arms of temporalis and masseter muscles varied substantially among muscle fibers. The values calculated from the centroid-to-centroid model were significantly different from those calculated using the ‘Distributed model’, which considered crucial 3D muscle attachment morphometry. Consequently, joint loading was underestimated by more than 50% in the centroid-to-centroid model. Therefore, it is necessary to consider 3D muscle attachment morphometry, especially for muscles with broad attachments, in TMJ musculoskeletal models to precisely quantify the joint mechanical environment critical for understanding TMJ function and mechanobiology.     

Fucose expression in skeletal muscle: a lectin histochemical study

Summary Binding sites for three fucose specific lectins, Aleuria aurantia agglutinin (AAA), Lotus tetragonolobus agglutinin (LTA) and Ulex europeus I agglutinin (UEA I), were investigated in sections from normal human and rat muscles, in muscle from patients with Duchenne muscular dystrophy (DMD) and in denervated and devascularized rat muscle. In normal human and rat muscle AAA detected fucosylated glycocompounds in the sarcoplasm, sarcolemma, interfibre connective tissue and vascular structures. In normal human muscle addition of fucose to the AAA incubation medium or treatment of the sections with formaldehyde followed by periodic oxidation before lectin incubation strongly inhibited the staining at all sites other than endothelial cells. In normal rat muscle the same staining procedures strongly inhibited the AAA binding at all sites other than the sarcolemma. Incubation with LTA resulted in a diffuse reaction around the vascular structures in rat muscle, while in human muscle a moderate, homogeneous staining was present in all muscle fibres. Treatment of the sections with formaldehyde and periodic acid before incubation with LTA resulted in strongly labelled muscle capillaries in both human and rat muscle. The only elements in the muscle tissues that were stained with UEA I were human endothelial cells. In denervated and devascularized rat muscle incubation with AAA revealed a novel fucose expression that appeared intracellularly in some necrotic fibres. The AAA-positive fucose residues in the sarcolemma of normal muscle fibres that were resistant to periodic acid oxidation could not be shown by AAA in denervated muscle. In DMD muscle a cryptic sarcolemmal fucose expression could be shown with AAA. It is suggested that both the sarcoplasm and sarcolemma of diseased muscle fibres show altered fucose expression.     

Oestrogen receptor β is present in both muscle fibres and endothelial cells within human skeletal muscle tissue

Oestrogen receptor β (ERβ) is expressed in human skeletal muscle tissue. In the present study, we have developed an immunohistochemical method to reveal if ERβ is located within the muscle fibres as well as within capillaries. Skeletal muscle biopsies were obtained from m. quadriceps femoris vastus lateralis in four healthy young subjects. Immunohistochemical triple staining was applied to transverse sections of paraffin-wax-embedded tissue. The basement membrane of muscle fibres and capillaries was identified by using an antibody to collagen IV, endothelial cells using an antibody to CD34 and ERβ using a corresponding antibody. The ERβ-positive (ERβ+) nuclei were located within the muscle fibre defined by the localisation of collagen IV. ERβ+ nuclei were also, for the first time, found in endothelial cells of capillaries in skeletal muscle tissue. Quantification was performed on transverse cryostat sections after performing a double staining (collagen IV and ERβ). It was shown that 24% of the ERβ+ nuclei were located within capillaries, and 76% were located within muscle fibres. In conclusion, ERβ in human skeletal muscle tissue is expressed not only in the muscle fibres themselves, but also within the capillary endothelial cells. This observation might improve understanding of the physiological role of oestrogen and its receptor.     

Identification of Functional Tumor Vessels by Resorcin-Crystal Violet Stain Infusion

A resorcin-crystal violet solution of low viscosity injected into the circulatory plexus supplying a tumor is used to identify and characterize functional tumor vessels. Unstained sections of tumor tissue demonstrate heavily stained vascular endothelium with no leakage of stain to extravascular tissue for intact vessels, and little or no background staining. The method is simple to apply for tumor vessel morphology and morphometry.     

Analysis of fibronectin expression during human muscle differentiation

Fibronectin expression during human muscle differentiation was investigated by determining its distribution in foetal, normal adult and dystrophic muscle and in foetal, normal adult and dystrophic muscle cultures during myogenesis. Muscle sections and muscle cultures were studied by indirect immunofluorescence staining using polyclonal and monoclonal anti-human antibodies. Mass and clonal muscle cultures were prepared from foetal, adult and dystrophic muscle tissue. Immunofluorescence staining detected fibronectin on the epimysium, perimysium and endomysium of transverse sections of normal adult muscle, while sarcoplasm was devoid of this glycoprotein. In foetal muscle, some fibers showed a prominent ring of fibronectin. In mass and clonal cultures, myoblasts were found to synthesize and accumulate fibronectin while myotubes did not. No difference in fibronectin distribution was observed between Duchenne Muscular Dystrophy (DMD) and control myotubes. An enzyme-linked immunoassay (ELISA), performed on homogenated muscle, sonicated fibroblasts and muscle cells, showed a high fibronectin level in fibroblasts when compared with the other samples tested.     

Retrieval of cryostat section for comparison of histochemistry and quantitative electron microscopy in a muscle fiber.

A method is presented that can be used to perform histochemical and morphometric analyses on the same muscle fiber. Freshly dissected fibers from medial gastrocnemius muscle of adult guinea pig were kept at a resting length and rapidly frozen. Serial frozen cross-sections were cut and reacted for myofibrillar adenosine triphosphatase and succinic dehydrogenase. The adjacent section, while still frozen, was immersed into 20 degrees C glutaraldehyde fixative to which EGTA was added to minimize artifactious contraction. The fixed section was processed for electron microscopy and the section rotated before thin sectioning to give longitudinal sections enabling study of sarcomeres. Ultrastructure was well-preserved despite slight disorganization of the contractile filaments and some vesiculation of the sarcoplasmic reticulum. The Z line width was measured and the mitochondrial volume fraction estimated by point counting morphometry from 89 fibers. The fibers with dark myofibrillar adenosine triphosphatase staining have Z widths of 547 +/- 165 A (n=69) and thoshosphatase staining have Z widths of 547 +/- 165 A (n=69) and those with light stain have 1023 +/- 113 A (n=20). The density of the succinic dehydrogenase reaction product in the fibers was divided into dark and light and the mitochondrial volume fractions were foud to be 4.3 +/- 2.1% (n=52) and 1.0 +/- 1.1% (n=37), respectively.     

Multi-step immunofluorescent analysis of vitamin D receptor loci and myosin heavy chain isoforms in human skeletal muscle

Vitamin D receptors have been shown to be present in human skeletal muscle using different techniques. We developed a multi-staining immunofluorescent method to detect vitamin D receptor expression and co-localize it with myosin heavy chain isoform expression in skeletal muscle biopsies in older female subjects. Serial sections were cut from frozen samples obtained by needle biopsy of the vastus lateralis. Samples were probed with a primary vitamin D receptor monoclonal antibody and then re-probed with a type IIa myosin heavy chain isoform-specific antibody. Independent unfixed sections followed a similar protocol and were probed with type IIx and type I myosin heavy chain isoform-specific antibodies. Immunohistochemistry and fluorescent microscopy co-localized vitamin D receptor loci and myosin heavy chain isoforms in whole skeletal muscle sections. We quantified intranuclear vitamin D receptor staining patterns and number of individual muscle fiber subtypes within a muscle section. Immunohistochemical staining of the vitamin D receptor was confirmed by Western blot using the same monoclonal antibody. This multi-staining immunofluorescent technique allows for measurement of intranuclear vitamin D receptor expression in the context of the specific muscle fiber type profile in a single section. This method can thus be a useful approach to study potential relationships between muscle fiber subtypes and vitamin D receptor expression.     

A new histochemical method for magnesium actomyosin adenosine triphosphatase at physiological pH

A new lead-precipitation technique for demonstrating magnesium-activated actomyosin adenosine triphosphatase (ATPase) at physiological pH and electrolyte levels in fixed skeletal muscle sections is reported. This method is compared with standard acid- and alkali-denatured muscle stained for calcium myosin ATPase as well as calcium-formalin denatured and pyrophosphate-formalin denatured muscle also stained for calcium myosin ATPase. The technique was developed using hamster skeletal muscle; however, it has also been applied to human, rat, and cat muscle. The fiber-type staining intensities of the formalin-denatured magnesium actomyosin ATPase closely resemble those of the formalin-denatured calcium myosin ATPase in rodents, but intensities in Type 1 fibers are reversed relative to calcium myosin ATPase in human muscle. Cat muscle shows intermediate characteristics.     

肿瘤核仁形成区形态定量诊断的误差及规范化分析

本文采用包括自动图象分析技术在内的ＡｇＮＯＲ形态定量学方法,以大肠肿瘤为模型,进行了ＡｇＮＯＲ定量形态学研究的误差分析,以探讨肿瘤ＡｇＮＯＲ定量诊断规范化的可能。结果表明,染色条件、视场目标选择和细胞计数量是引起ＡｇＮＯＲ定量诊断的主要误差;恒定染色环境,正确选择欲测细胞及测定足够量的细胞是使ＡｇＮＯＲ形态定量诊断规范化的途径。     

Specific cytochemical stains in the image analysis of subcellular organelles

This paper describes our work concerning densitometry and morphometry of subcellular structures in thin sections. The techniques of automatic image analysis were applied to light and electron microscopic observations of enzymatically stained lysosomes, renal brush borders and mitochondria (in human and rat kidney) and peroxisomes (in human liver). To obtain significant measurements of the enzymatic activity, specific staining techniques were developed and applied, including an improved staining of acid phosphatase for lysosomes. Optical densities were obtained by videodensitometry and electron densities of peroxisomes were obtained by digitizing and processing scanning transmission electron microscopic images. In subsequent steps, delineations and parameter estimation are performed by software. Included was an examination of delineation techniques, which showed improved results from the use of a newly developed local boundary search algorithm. The combination of these techniques was used to study changes in peroxisome and lysosome compartment in liver and kidney, some results of which are also reported.     

Application of nile blue and nile red, two fluorescent probes, for detection of lipid droplets in human skeletal muscle

Using frozen sections from human muscle biopsies, we assessed the value of Nile blue and Nile red, two fluorescent probes, as stains for lipid droplets in normal and pathological skeletal muscle fibers. In normal muscle, lipid storage disorders, and mitochondrial myopathies, Nile blue stained the lipid droplets as yellow-gold fluorescent structures. The lipid droplets were also seen as yellow-gold fluorescent structures in Nile red-stained sections, but the outstanding feature in these preparations was the staining of the membrane network of the muscle fibers and membrane proliferations in pathological muscle as red-orange fluorescent structures. These results suggest that both Nile blue and Nile red stains are useful for visualization of lipid droplets and membrane proliferations in pathological muscle biopsies.     

假肥大型肌营养不良肌细胞抗肌萎缩蛋白的免疫组化研究

目的研究假肥大型肌营养不良患者肌细胞中抗肌萎缩蛋白(dystrophin)的表达及其诊断意义。方法应用针吸型活检术取121例假肥大型肌营养不良症患者(108例DMD患者,13例BMD患者)的肌组织,采用HE染色观察被检肌肉病理改变,免疫组织化学染色技术检测抗肌营养不良蛋白表达,以正常人肌细胞作为对照。结果正常人肌细胞膜上抗肌营养不良蛋白染色阳性,呈完整环形条带沿肌细胞膜分布;DMD患者肌膜完全无显色;BMD患者染色弱阳性,可见沿肌细胞膜分布的间断表达。结果 应用针吸型活检技术和免疫组化染色法检测抗肌营养不良蛋白,有助于DMD和BMD确诊及鉴别诊断。     

Reproducibility of morphometric measurements of amyloid after various staining methods

Four histologic staining methods used for detecting amyloid (Congo red, viewed in both normal and polarized light, Sirius red, Crystal violet and Thioflavine T) were applied to heart muscle autopsy samples from 19 patients who suffered from amyloidosis. The amount of amyloid present was evaluated with morphometry (point counting) by five pathologists, and the interobserver reproducibility and variation of point counting in these staining methods were analyzed. The Sirius red method showed the least variation and was the most suitable stain for demonstrating amyloid with respect to reproducibility. Thioflavine T showed the greatest variation and was the least suitable stain with respect to reproducibility. The range of variation was considerable in all staining methods. The results show that stains differ in their specificity and sensitivity in staining amyloid, observers differ in their interpretation of staining results and certain stains result in more uniform interpretations than do others.     

Staining of Different Tissues in Thick Epoxy Resin-Impregnated Sections of Human Fetuses

Sections of undecalcified human fetuses, fixed in formaldehyde, embedded in the epoxy resin Biodur E 12 and cut on a diamond-wire saw were stained according to a slight modification of the method described by Laczko and Levai. The sections were immersed in a methylene blue/azure II solution at 90 C for at least 3 min and counterstained with a basic fuchsin solution at the same temperature. Differential staining was as follows: bone stained pinkish; cartilage, violet; collagen fibers, blue-violet; elastic fibers, red and muscle fibers, green-blue. Most other tissues were stained blue-violet against the transparent background of the embedding epoxy resin. Thanks to the distinct and differential staining of each tissue, contrast is sufficient for black and white as well as for color photography.     

CYTOCHEMICAL LOCALIZATION OF LACTIC DEHYDROGENASE IN WHITE SKELETAL MUSCLE

The limitations of the conventional histochemical methods for localization of lactic dehydrogenase (LDH) in white skeletal muscle have been analyzed quantitatively. It is demonstrated that more than 80 per cent of LDH diffuses into the incubation medium within the first 10 minutes of incubation. Furthermore, it is confirmed that the addition of phenazine methosulfate (PMS) to the ingredients of the histochemical reaction for LDH increases substantially the capacity of the white muscle extract to reduce Nitro-BT. Based on these observations, a modified method of cytochemical localization of LDH has been developed. This method prevents the leakage of LDH from tissue sections by the application of all the ingredients of the histochemical reaction to tissue sections in a thin gelatin film. The incubation mixture contains PMS so that the staining system is independent of tissue diaphorase. The application of this method to the adductor magnus muscle of the rabbit revealed a fine reticulum in the sarcoplasm of all muscle fibers, in addition to the staining of mitochondria. The distribution of the staining suggests that LDH is localized in the sarcoplasmic reticulum.     

A method for myoglobin in cryostat sections of muscle by precipitation with sulfosalicylic acid.

Transverse cryostat sections of skeletal muscle were fixed in a solution containing 1.5% glutaraldehyde and 1.5% sulfosalicylic acid and stained in a solution containing equal volumes of 3% hydrogen peroxide and 50% ethanol saturated with o-tolidine. Myoglobin in the sarcoplasm of muscle fibers was precipitated and stained blue. Applicability of this method to cryostat sections, without glutaraldehyde fixations prior to freezing, allowed the myoglobin content of individual muscle fibers to be correlated with other histochemical characteristics of the same fibers seen in serial sections. In the dark red bovine sternomandibularis muscle, fibers with weak adenosine triphosphatase (ATPase) and strong succinate dehydrogenase (SDH) activity always exhibited strong myoglobin staining. An equal degree of staining was found in many fibers with strong ATPase and intermediate to strong SDH activity. Fibers with strong ATPase and weak SDH activity were less strongly stained than the preceding types.     

A Method for Myoglobin in Cryostat Sections of Muscle by Precipitation with Sulfosalicylic Acid

Transverse cryostat sections of skeletal muscle were fixed in a solution containing 1.5% glutaraldehyde and 1.5% sulfosalicylic acid and stained in a solution containing equal volumes of 3% hydrogen peroxide and 50% ethanol saturated with o-tolidine. Myoglobin in the sarcoplasm of muscle fibers was precipitated and stained blue. Applicability of this method to cryostat sections, without glutaraldehyde fixation prior to freezing, allowed the myoglobin content of individual muscle fibers to be correlated with other histochemical characteristics of the same fibers seen in serial sections. In the dark red bovine sternomandibularis muscle, fibers with weak adenosine triphosphatase (ATPase) and strong succinate dehydrogenase (SDH) activity always exhibited strong myoglobin staining. An equal degree of staining was found in many fibers with strong ATPase and intermediate to strong SDH activity. Fibers with strong ATPase and weak SDH activity were less strongly stained than the preceding types.     

Staining of different tissues in thick epoxy resin-impregnated sections of human fetuses

Sections of undecalcified human fetuses, fixed in formaldehyde, embedded in the epoxy resin Biodur E 12 and cut on a diamond-wire saw were stained according to a slight modification of the method described by Laczkó and Lévai. The sections were immersed in a methylene blue/azure II solution at 90 C for at least 3 min and counterstained with a basic fuchsin solution at the same temperature. Differential staining was as follows: bone stained pinkish; cartilage, violet; collagen fibers, blue-violet; elastic fibers, red and muscle fibers, green-blue. Most other tissues were stained blue-violet against the transparent background of the embedding epoxy resin. Thanks to the distinct and differential staining of each tissue, contrast is sufficient for black and white as well as for color photography.     

A monoclonal antibody against cytochrome c oxidase distinguishes cardiac and skeletal muscle mitochondria

The mitochondrial enzyme cytochrome c oxidase (COX) in eukaryotes consists of at least seven subunits, three of which (I-III) are encoded by mitochondrial DNA (mitDNA) and the others (IV-VII) by the nuclear genome. There is increasing evidence that COX in mammals exists in multiple tissue-specific forms, presumably specified by nuclearly encoded subunits. We performed immunologic studies in human cardiac and skeletal muscle, using a monoclonal antibody raised against subunit IV of COX purified from human cardiac muscle. In immunotitration studies, the antibody bound with high affinity to mitochondria from cardiac muscle, but reacted only weakly with mitochondria from skeletal muscle. Similarly, immunocytochemical studies showed prominent mitochondrial staining in frozen sections of heart, but no staining in sections of mature skeletal muscle. Although this antibody did not stain mitochondria in mature skeletal muscle, it clearly stained mitochondria in myoblasts and immature myotubes of human muscle cultures, suggesting that mitochondria in immature muscle cells are different from those in mature muscle, and similar to heart mitochondria. Immunotitration data using either native or denatured COX protein from heart or skeletal muscle showed similar immunoreactivity. These studies indicate that the epitope for recognition by this antibody is exposed in mitochondria from heart and immature muscle cells, but masked in mitochondria from mature skeletal muscle.