A Revised Method for the “Quad” Stain

Since the original publication of this staining method (Stain Techn., 18, 95), various improvements have been made. These are chiefly in the formula for acid alizarine blue, Solution 2, given below and the composition of Solution 4. The author substituted ammonia alum for aluminum sulphate, and Richard C. Webster of our department did the work with the acetic-acid-sodium-acetate buffer. He found that the final staining solution of pH 2.9 gave the sharpest and bluest nuclei and clearest transparent and contrasting cytoplasm of a pinkish hue. With our previous formula, muscle fibrils often stained so deeply that they detracted from the value of the slides, made them too opaque and obscured the nuclei. This has now been overcome. This also allows the elastic tissue to show better and to contrast with muscle fibers. This is especially noted in studies of the heart and blood vessels.     

Improved Histology for the Chick-Quail Chimera

A simple modification of nuclear staining after acid hydrolysis has been made which provides easy identification of quail nuclear markings in a chick-quail chimera. This method also improves the histologic detail normally seen with hematoxylin and eosin when compared to the more commonly used Feulgen reaction. Embryonic tissues can be fixed in Zenker's or Helly's solution and the sections obtained are hydrolyzed in acid (3.5 N HCl at 37 C for 40-50 min). After acid hydrolysis the sections are stained with hematoxylin and eosin rather than Schiff reagent and fast green. The interphase nuclei of chick cells show homogeneous or mottled purplish blue staining, while quail nuclei contain a dark blue spot. This staining corresponds to the reddish purple staining of the quail's heterochromatin seen adjacent to the nucleolus in the standard Feulgen stain. This new technique facilitates identification of quail cell types in the chick host and provides superior histology of the chick tissues by demonstrating cytoplasmic detail.     

Morphological analysis of rat ureteric terminal arterioles in situ

Confocal imaging of Fluo‐4, Propidium iodide, and di‐8‐Anepps loaded ureter were used to study the morphology of terminal arterioles with an inner diameter <50 μm in intact rat ureter. Optical sectioning showed that the muscle coat of the terminal arterioles consisted of a monolayer of highly curved smooth muscle cells which run circumferentially around the endothelium. This technique allowed not only to measure the inner diameter of the terminal arterioles but also to define the orientation and number of revolutions an individual smooth muscle cell made around the endothelium. We measured thickness, width, length, and morphological profile of the myocytes and endothelial cells. Propidium iodide staining showed nuclei of individual cells by continuous imaging at high resolution in serial optical sections. Conventional haematoxylin‐eosin, Masson's tri‐chrome staining, and transmission electron microscopy were also used in this study to compare the measurements obtained from live confocal imaging with histological standard methods. Parameters obtained from live imaging were significantly different. This technique of live staining allowed measuring the cellular and nuclear dimensions of the terminal arterioles in their natural environment which are important in studying the effects of vascular disease or aging on vascular structure. J. Morphol. 2013. © 2013 Wiley Periodicals, Inc.     

比格犬卵巢子宫内雌激素受体ERα、ERβ的免疫组织化学定位

目的研究雌激素受体α,β在比格犬卵巢及子宫内的定位。方法采用免疫组化SP法DAB显色结合BCIP/NBT及AEC显色检测ERα、ERβ在比格犬子宫及卵巢内的表达。结果比格犬ERα主要表达于卵泡颗粒细胞、卵巢间质腺腺上皮细胞及子宫内膜腺体腺上皮细胞胞核内,胞质内仅有少量表达,而在卵泡膜内膜的间质细胞,腺体周围的基质细胞及小动脉血管内皮细胞和平滑肌细胞、小静脉内皮细胞的胞核内有少量表达。而ERβ则以相同的组织特异性主要表达于上述组织细胞的胞质内,在胞核内有微弱表达。ERα表达于膜黄体细胞的胞核内,而在黄体颗粒细胞胞核与胞质内均有表达。而ERβ则仍特异表达于不同生理阶段黄体细胞的胞质内。BCIP/NBT与AEC双染未见ERα、ERβ在子宫内有明显的共表达现象。结论比格犬ERα、ERβ在子宫与卵巢组织内定位不同,ERα主要定位于胞核,在胞质内有微弱表达,而ERβ主要定位于胞质,在胞核内有零星表达。     

Sarcopenia is not due to lack of regenerative drive in senescent skeletal muscle

Sarcopenia, loss of skeletal muscle mass, is a hallmark of aging commonly attributed to a decreased capacity to maintain muscle tissue in senescence, yet the mechanism behind the muscle wasting remains unresolved. To address these issues we have explored a rodent model of sarcopenia and age-related sensorimotor impairment, allowing us to discriminate between successfully and unsuccessfully aged cohort members. Immunohistochemistry and staining of cell nuclei revealed that senescent muscle has an increased density of cell nuclei, occurrence of aberrant fibers and fibers expressing embryonic myosin. Using real-time PCR we extend the findings of increased myogenic regulatory factor mRNA to show that very high levels are found in unsuccessfully aged cohort members. This pattern is also reflected in the number of embryonic myosin-positive fibers, which increase with the degree of sarcopenia. In addition, we confirm that there is no local down-regulation of IGF-I and IGF-IR mRNA in aged muscle tissue; on the contrary, the most sarcopenic individuals showed significantly higher local expression of IGF-I mRNA. Combined, our results show that the initial drive to regenerate myofibers is most marked in cases with the most advanced loss of muscle mass, a pattern that may have its origin in differences in the rate of tissue deterioration and/or that regenerating myofibers in these cases fail to mature into functional fibers. Importantly, the genetic background is a determinant of the pace of progression of sarcopenia.     

Morphogenesis of transverse straited muscles

During the past 50 years there have been significant advances in our understanding of striated muscle development, both from tissue culture studies and from observations of myogenesis in the developing foetus. Experiments, using the distinctive nucleolar marker of quail nuclei, have led to reexamination of the source of the body's muscle masses. Advances have been made in our understanding of the events which occur and mechanisms involved in the formation of multinucleated myofibers from mononucleated myoblasts. Recently, the development of entire muscle organs has been studied. The present review attempts to synthesize the results of both in vivo and in vitro studies of myogenesis, comparing both their similarities and their differences. Attention has been focused on recent advances in our understanding of the source of muscles in the intact foetus, the formation of extrafusal and intrafusal fibers, and the development of the afferent and efferent neuromuscular relationships.     

Improved immunocytochemical detection of daunomycin

Improved immunocytochemical (ICC) detection of the anthracycline anticancer antibiotic daunomycin (DM) has been achieved by use of hydrogen peroxide oxidation prior to ICC staining for DM. The new method greatly enhanced the localization of DM accumulation in cardiac, smooth and skeletal muscle of rats after a single i.v. dose of the drug. DM accumulated in the nuclei as well as in the sarcoplasm, where it occurred in the form of small granules, which were particularly evident in cardiac muscle cells. The distribution of the granules coincided with that of mitochondria. Uptake of DM in nuclei and mitochondria of heart muscle cells may help to improve our understanding of the cardiac toxicity of DM and related anthracyclin antibiotics. A number of ELISA tests were carried out in order to elucidate the mechanims of H₂O₂−assisted antigen retrieval. A possible mechanism is that DM is reduced and converted to its semiquinone and/or hydroquinone derivative in vivo. Oxidation by hydrogen peroxide acts to convert these derivatives back to the native antigen. The improved ICC methodology using oxidation to recreate native antigens from reduced metabolites may be helpful also with respect to the localization of other drugs.     

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.     

Satellite cell proliferation in low frequency-stimulated fast muscle of hypothyroid rat

Satellite cell proliferation was assessed inlow-frequency-stimulated hypothyroid rat fast-twitch muscle by5-bromo-2'-deoxyuridine (BrdU) labeling and subsequent staining oflabeled muscle nuclei, and by staining for proliferating cell nuclearantigen (PCNA). BrdU labeling and PCNA staining were highly correlatedand increased approximately fourfold at 5 days of stimulation, decayedthereafter, but remained elevated over control in 10- and 20-daystimulated muscles. Myogenin mRNA was ~4-fold elevated at 5 days and1.5-fold at 10 days. Staining for myogenin protein yielded resultssimilar to that for PCNA and BrdU. Furthermore, a detailed examination of the pattern of myogenin staining revealed that the number of myogenin-positive nuclei was elevated in the fast pure IIB fiber population at 5 and 10 days of chronic low-frequencystimulation. By 20 days, myogenin staining was observed intransforming fast fibers that coexpressed embryonic and adult myosinheavy chain isoforms. In the slower fiber populations (i.e., IIA andI), myogenin-positive transforming fibers that coexpressed embryonicmyosin heavy chain, appeared already at 5 days. Thus the satellite cellprogeny on slower fibers seemed to proliferate less and to fuse earlierto their associated fibers than the satellite cell progeny on fast fibers. We suggest that the increase in muscle nuclei of the fast fibers might be a prerequisite for fast-to-slow fiber type transitions.

     

Techniques for staining fungal nuclei and appendages

PUNITHALINGAM, E., 1989. Techniques for staining fungal nuclei and appendages. The use of HCl-Giemsa stain for staining nuclei and a modified Leifson's flagellum staining technique for staining appendages in Coelomycetes has produced useful information which could further our knowledge of fungi and help to reappraise earlier concepts.     

Quantitative Segmentation of Fluorescence Microscopy Images of Heterogeneous Tissue: Application to the Detection of Residual Disease in Tumor Margins

Purpose

To develop a robust tool for quantitative in situ pathology that allows visualization of heterogeneous tissue morphology and segmentation and quantification of image features.

Materials and Methods

Tissue excised from a genetically engineered mouse model of sarcoma was imaged using a subcellular resolution microendoscope after topical application of a fluorescent anatomical contrast agent: acriflavine. An algorithm based on sparse component analysis (SCA) and the circle transform (CT) was developed for image segmentation and quantification of distinct tissue types. The accuracy of our approach was quantified through simulations of tumor and muscle images. Specifically, tumor, muscle, and tumor+muscle tissue images were simulated because these tissue types were most commonly observed in sarcoma margins. Simulations were based on tissue characteristics observed in pathology slides. The potential clinical utility of our approach was evaluated by imaging excised margins and the tumor bed in a cohort of mice after surgical resection of sarcoma.

Results

Simulation experiments revealed that SCA+CT achieved the lowest errors for larger nuclear sizes and for higher contrast ratios (nuclei intensity/background intensity). For imaging of tumor margins, SCA+CT effectively isolated nuclei from tumor, muscle, adipose, and tumor+muscle tissue types. Differences in density were correctly identified with SCA+CT in a cohort of ex vivo and in vivo images, thus illustrating the diagnostic potential of our approach.

Conclusion

The combination of a subcellular-resolution microendoscope, acriflavine staining, and SCA+CT can be used to accurately isolate nuclei and quantify their density in anatomical images of heterogeneous tissue.     

A Fluorescence Staining-Methyl Salicylate Clearing Technique for Demonstrating Pollen Nuclei

Recently several DNA-binding fluotochromes have been used for demonstrating pollennuclei. However, the autofluorescence of pollen wall often obscured the fluorescence of nuclei, thus limited the use of this method. Methyl salicylate (MS) as a clearing agent has shownexcellent effect for observing embryo sac in whole-mounted ovules. This aroused me to trya combination of fluorescent staining with MS clearing in orded to make a better demonstration of the pollen nuclei. Mature 2-celled or 3-celled pollen of several angiosperm species stained with Hoechst 33258(H33258) and cleared (via ethanol dehydration) with MS showed clearcut fluorescence oftheir generative or sperm nuclei and vegetative nucleus. MS greatly decreased the wall fluorescence and increased the transparency of the pollen contents, meanwhile maintained the H33258stained fluorescence, consequently made the nuclei brighter under a darkened background. For example, in sunflower pollen a pair of elongated and winding sperm nuclei whichcould not be identified after simple H33258 staining were quite visible after MS clearing, inartificially germinated pollen tubes, the locomotion of nuclei from pollen grain into the tube,the sequence of generative and vegetative nucle travelling along the tube and the division of generative nucleus into two sperm nuclei could be well followed by this method. The present technique may be adoptable for observations on the processes of microsporogenesis and male gametophyte development, and rogenesis in cultured anthers, and also possiblyfor tracing the nuclear events during pollination-fertilization.     

An improved method to investigate staining kinetics in single cells

Summary A new method to analyze staining processes in single cells of histochemical and cytochemical specimens in situ is described. The combination of a microscope photometer with a perfusion cuvette developed in our laboratory allows the continuous observation of a cell during the staining process. The flow rate dependence of the staining process has been examined demonstrating the strong suppression of the diffusional boundary layer adjacent to the cell surface by sufficiently high flow rates. Experiments to find optimal conditions for the kinetic analysis of the staining reaction of nuclei in lymphocytes, neutrophile granulocytes and monkey kidney cells with thionin are described. Half-staining times of the binding of monomer dye molecules and aggregates to nuclei have been calculated; they depend on the pretreatment of the cells. The addition of electrolytes decreases the rate of staining. The formation of aggregates obeys approximately a first-order reaction law and the binding of monomers provides an order of reaction of n=0.5.     

Immunocytochemical detection of enhanced expression of c-myc protein in the heart of cardiomyopathic hamster

An immunocytochemical study was performed to examine the expression of cellular c-myc protein in the heart of 30-, 120- and 180-day-old cardiomyopathic Syrian UM-X7.1 hamsters. The heart of age- and sex-matched BIO-RB hamster was used as normal control. In paraffin sections, an immunostaining for c-myc was markedly increased in cytoplasm of cells from the UM-X7.1 heart as compared with that of the BIO-RB heart which showed a weak staining. However, c-myc was localized in nuclei of cells in frozen sections of the heart. Specific cell types of the heart were differentiated with anti-vimentin, and we found that the increased expression of c-myc was present in nuclei of muscle cells of the UM-X7.1 myocardium. A quantitative study of c-myc-positive nuclei of muscle and nonmuscle cells was carried out by a video micrometer. The mean number of c-myc-positive nuclei of muscle cells was significantly higher in the cardiomyopathic heart than in the control heart from hamsters of all ages studied. These results suggest that the increase of c-myc protein may relate to the pathological state or pathogenesis of the hereditary cardiomyopathy.     

An improved method to investigate staining kinetics in single cells

A new method to analyze staining processes in single cells of histochemical and cytochemical specimens in situ is described. The combination of a microscope photometer with a perfusion cuvette developed in our laboratory allows the continuous observation of a cell during the staining process. The flow rate dependence of the staining process has been examined demonstrating the strong suppression of the diffusional boundary layer adjacent to the cell surface by sufficiently high flow rates. Experiments to find optimal conditions for the kinetic analysis of the staining reaction of nuclei in lymphocytes, neutrophile granulocytes and monkey kidney cells with thionin are described. Half-staining times of the binding of monomer dye molecules and aggregates to nuclei have been calculated; they depend on the pretreatment of the cells. The addition of electrolytes decreases the rate of staining. The formation of aggregates obeys approximately a first-order reaction law and the binding of monomers provides an order of reaction of n = 0.5.     

Identification of nuclei from apoptotic, necrotic, and viable lymphoid cells by using multiparameter flow cytometry.

BACKGROUND: Methods widely used to detect apoptosis do not allow us to easily distinguish between nuclei from viable or necrotic cells. Even if apoptosis and necrosis seem to occur as alternatives at the single cell level, they could be present simultaneously in a cell population much more frequently than expected. For this reason, attention was focused on attempting to recognize, by multiparameter flow cytometry, the characteristics of viable cells and of apoptotic or necrotic dead cells. METHODS: Apoptosis and necrosis were induced in vitro in murine thymocytes and lymphocytes from adult peripheral blood by using dexamethasone or prostaglandin E2 treatment and heat shock at 60 degrees C or hydrogen peroxide, respectively. Traditional methods, such as DNA gel electrophoresis and propidium iodide staining followed by single-fluorescence analysis or annexin-V-fluorescein isothiocyanate plus propidium iodide staining by using flow cytometry, were compared with a new method. This method consisted of combined light-scatter and red fluorescence analysis by flow cytometry after isolation of nuclei by hypotonic solution as well as high-dose detergent treatment and DNA staining with propidium iodide. RESULTS: Results showed that, although traditional methods such as DNA-gel electrophoresis and single-parameter fluorescence flow cytometry analysis were unable, as expected, to discriminate among viability, apoptosis, and necrosis, our new method has enabled us to easily identify nuclei from viable, apoptotic, and necrotic cells. Results obtained by using our method were comparable to those obtained by using two-color analysis of cells after propidium iodide/annexin V staining. CONCLUSIONS: A highly reproducible, inexpensive, rapid, and easily accessible method of analysis has been developed for simultaneously detecting apoptosis and necro sis.     

Immunohistochemical Differentiation of Fiber Types in Human Skeletal Muscle Using Monoclonal Antibodies to Slow and Fast Isoforms of Troponin I Subunit

The cDNA sequence of troponin I (TnI), one of the subunits of the skeletal muscle regulatory protein, differs between slow-twitch muscle and fast-twitch muscle. We prepared monoclonal antibodies td the slow and fast isoforms of human TnI for the purpose of differentiating muscle fiber types in human neuromuscular disorders. Slow TnI antibody was labeled with tetramethylrhodamine isothiocyanate while fast TnI antibody was labeled with fluorescein isothiocyanate; then these two antibodies were mixed. This mixture was then used to stain biopsied muscle from patients with neuromuscular disorders. It was possible to differentiate muscle fibers into slow, fast and intermediate fibers having various contents of slow and fast TnI. In tissue composed of small muscle fibers, this method facilitated differentiation of types of muscle fibers by allowing staining of only a single section. The usefulness of our technique using slow and fast TnI antibodies is discussed in comparison with ATPase staining. Because our staining method can distinguish slow and fast fiber components, it is useful for clinical application.     

Analysis of a 43-kDa glycoprotein from the intracellular parasitic nematode Trichinella spiralis.

The L1 larvae of the parasitic nematode Trichinella spiralis invade skeletal muscle and initiate a process that has been interpreted to represent skeletal muscle dedifferentiation. In this process, the infected region of the muscle cell is converted into a unique structure, called the Nurse cell. The nematode T. spiralis can survive for tens of years within the cytoplasm of the Nurse cell and secretes proteins into the cytoplasm that are believed to play a role in mediating the Nurse cell formation or maintenance. We have cloned a cDNA encoding the T. spiralis-derived, 43-kDa secreted protein. Structural analysis of the predicted 344-amino acid sequence revealed an N terminally located signal peptide and a potential helix-loop-helix motif in the main body of the protein. Antibodies raised against the 43-kDa recombinant protein were used in immunocytolocalizations of T. spiralis-infected skeletal muscle sections. These antibodies strongly stained the Nurse cell nuclei and the nematode itself. Specific, though slightly weaker staining also occurred in the Nurse cell cytoplasm. In Western blots, the antibodies react with the 43-kDa protein but also detected at least two other T. spiralis-secreted proteins. DNA hybridizations reveal at least one additional 43-kDa-related sequence encoded in the T. spiralis genome. We conclude that either the 43-kDa protein and/or a closely related 43-kDa family member is secreted into the muscle and translocates to the muscle-derived nuclei. This model may provide insights into the mechanisms involved in Nurse cell formation.