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  • Unobstructed Multiscale Imaging of Tissue Sections for Ultrastructural Pathology Analysis by Backscattered Electron Scanning Microscopy
    J. Histochem. Cytochem. (IF 2.37) Pub Date : 2019-08-06
    Mike Reichelt; Meredith Sagolla; Anand K. Katakam; Joshua D. Webster

    Ultrastructural analysis of healthy, diseased, or experimental tissues is essential in diagnostic and investigative pathology. Evaluation of large tissue areas with suborganelle resolution is challenging because biological structures ranging from several millimeters to nanometers in size need to be identified and imaged while maintaining context over multiple scales. Imaging with field emission scanning electron microscopes (FE-SEMs) is uniquely suited for this task. We describe an efficient workflow for the preparation and unobstructed multiscale imaging of tissue sections with backscattered electron scanning electron microscopy (BSE-SEM) for applications in ultrastructural pathology. We demonstrate that a diverse range of tissues, processed by conventional electron microscopy protocols and avoiding the use of mordanting agents, can be imaged on standard glass slides over multiple scales, from the histological to the ultrastructural level, without any visual obstructions. Our workflow takes advantage of the very large scan fields possible with modern FE-SEMs that allow for the acquisition of wide-field overview images which can be explored at the ultrastructural level by digitally zooming into the images. Examples from applications in pulmonary research and neuropathology demonstrate the versatility and efficiency of this method. This BSE-SEM-based multiscale imaging procedure promises to substantially simplify and accelerate ultrastructural tissue analysis in pathology.

    更新日期:2019-12-25
  • MiR-210 Is Overexpressed in Tumor-infiltrating Plasma Cells in Triple-negative Breast Cancer
    J. Histochem. Cytochem. (IF 2.37) Pub Date : 2019-12-01
    Isabelle Bar; Ivan Theate; Sandy Haussy; Gabriela Beniuga; Javier Carrasco; Jean-Luc Canon; Paul Delrée; Ahmad Merhi

    Triple-negative breast cancer (TNBC) is a heterogeneous group of breast cancer and is characterized by aggressiveness and poor prognosis. MicroRNA represents a new class of biomarkers, and accumulating evidence indicates that microRNAs contribute to tumorigenesis and cancer metastasis. It has been described that miR-210 is highly expressed in TNBC, and its overexpression had been linked to poor prognosis. In a previous work, we showed that in TNBC miR-210 is expressed in tumor cells and also in the tumor microenvironment (TME), particularly in inflammatory CD45-LCA positive cells. However, the exact identity of these cells remained unknown. In this study, we performed in situ hybridization and immunohistochemistry using validated antibodies for the different specific immune cell markers on adjacent sections of 23 TNBC infiltrated with immune cells. We found that miR-210 expressing cells in the TME were stained positive with CD79a, a B-cell lineage marker. These tumor-infiltrating cells were negative for CD20 and Ki-67 but positive for MUM1 and CD38 and also expressed immunoglobulins, indicating that they are immunoglobulin-producing plasma cells (PCs). To the best of our knowledge, this is the first study demonstrating miR-210 expression in tumor-infiltrating PCs.

    更新日期:2019-12-25
  • Similarities Between Stem Cell Niches in Glioblastoma and Bone Marrow: Rays of Hope for Novel Treatment Strategies
    J. Histochem. Cytochem. (IF 2.37) Pub Date : 2019-09-30
    Vashendriya V.V. Hira; Barbara Breznik; Miloš Vittori; Annique Loncq de Jong; Jernej Mlakar; Roelof-Jan Oostra; Mohammed Khurshed; Remco J. Molenaar; Tamara Lah; Cornelis J.F. Van Noorden

    Glioblastoma is the most aggressive primary brain tumor with poor patient survival, which is at least partly due to a small subpopulation of glioblastoma cells, glioblastoma stem cells (GSCs). GSCs are localized in niches where they are maintained as slowly dividing cells, which results in resistance to therapy.1–5 It has been hypothesized that forcing GSCs out of their protective niches has therapeutic potential, because it sensitizes GSCs to irradiation and chemotherapy. Understanding of the functioning of GSC niches, that is, molecular mechanisms that retain GSCs in these protective niches, is required for the development of novel therapeutic strategies targeted against GSCs.

    更新日期:2019-12-25
  • Localization of Tricellular Tight Junction Molecule LSR at Midbody and Centrosome During Cytokinesis in Human Epithelial Cells
    J. Histochem. Cytochem. (IF 2.37) Pub Date : 2019-10-29
    Takumi Konno; Takayuki Kohno; Shin Kikuchi; Hiroshi Shimada; Seiro Satohisa; Kenichi Takano; Tsuyoshi Saito; Takashi Kojima

    Epithelial integrity and barrier function are maintained during cytokinesis in vertebrate epithelial tissues. The changes in localization and the roles of tricellular tight junction molecule lipolysis-stimulated lipoprotein receptor (LSR) during cytokinesis are not well known, although new tricellular tight junctions form at the flank of the midbody during cytokinesis. In this study, we investigated the changes in localization and the role of LSR at the midbody and centrosome during cytokinesis using human endometrial carcinoma cell line Sawano, comparing the tricellular tight junction molecule tricellulin; bicellular tight junction molecules occludin, claudin-7, zonula occludens-1, and cingulin; and the epithelial polarized related molecules apoptosis-stimulating of p53 protein 2, PAR3, and yes-associated protein. During cytokinesis induced by treatment with taxol, the epithelial barrier was maintained and the tricellular tight junction molecules LSR and tricellulin were concentrated at the flank of the acetylated tubulin–positive midbody and in γ-tubulin-positive centrosomes with the dynein adaptor Hook2, whereas the other molecules were localized there as well. All the molecules disappeared by knockdown using small interfering RNAs. Furthermore, by the knockdown of Hook2, the epithelial barrier was maintained and most of the molecules disappeared from the centrosome. These findings suggest that LSR may play crucial roles not only in barrier function but also in cytokinesis.

    更新日期:2019-12-25
  • Tissue-specific Fixation Methods Are Required for Optimal In Situ Visualization of Hyaluronan in the Ovary, Kidney, and Liver
    J. Histochem. Cytochem. (IF 2.37) Pub Date : 2019-11-12
    Jennifer E. Rowley; Gillian E. Rubenstein; Sharrόn L. Manuel; Natalie L. Johnson; Jordan Surgnier; Pinelopi P. Kapitsinou; Francesca E. Duncan; Michele T. Pritchard

    Hyaluronan (HA) is a ubiquitous component of the extracellular matrix (ECM) in vertebrate tissues.1,2 HA is an unbranched, non-sulfated glycosaminoglycan consisting of repeating glucuronic acid and N-acetylglucosamine units, and has numerous distinct biological functions. Through associations with other ECM components, such as proteoglycans, HA provides structural organization and integrity to the ECM.3 At physiological pH, HA is highly anionic and thus hydrophilic. As a result, HA can bind to water up to 1000-fold its own weight. Thus, HA is critical for hydration and lubrication in tissues, particularly soft connective tissues and joints.4 In addition to its structural role, HA is also a potent signaling molecule and mediates cell motility, adhesion, and proliferation.5–7

    更新日期:2019-12-25
  • Localization of Tfap2β, Casq2, Penk, Zic1, and Zic3 Expression in the Developing Retina, Muscle, and Sclera of the Embryonic Mouse Eye
    J. Histochem. Cytochem. (IF 2.37) Pub Date : 2019-10-22
    Yong Wan; Casey White; Nadine Robert; Matthew B. Rogers; Heather L. Szabo-Rogers

    Optic development involves sequential interactions between several different tissue types, including the overlying ectoderm, adjacent mesoderm, and neural crest mesenchyme and the neuroectoderm. In an ongoing expression screen, we identified that Tfap2β, Casq2, Penk, Zic1, and Zic3 are expressed in unique cell types in and around the developing eye. Tfap2β, Zic1, and Zic3 are transcription factors, Casq2 is a calcium binding protein and Penk is a neurotransmitter. Tfap2β, Zic1, and Zic3 have reported roles in brain and craniofacial development, while Casq2 and Penk have unknown roles. These five genes are expressed in the major tissue types in the eye, including the muscles, nerves, cornea, and sclera. Penk expression is found in the sclera and perichondrium. At E12.5 and E15.5, the extra-ocular muscles express Casq2, the entire neural retina expresses Zic1, and Zic3 is expressed in the optic disk and lip of the optic cup. The expression of Tfap2β expanded from corneal epithelium to the neural retina between E12.5 to E15.5. These genes are expressed in similar domains as Hedgehog (Gli1, and Ptch1) and the Wnt (Lef1) pathways. The expression patterns of these five genes warrant further study to determine their role in eye morphogenesis:

    更新日期:2019-11-27
  • Beclin 1 Interacts With Active Caspase-3 and Bax in Oocytes From Atretic Follicles in the Rat Ovary
    J. Histochem. Cytochem. (IF 2.37) Pub Date : 2019-10-04
    María L. Escobar; Olga M. Echeverria; Sebastián Palacios-Martínez; Silvia Juárez-Chavero; Luis Sánchez-Sánchez; Gerardo H. Vázquez-Nin

    Oocyte cell death is a normal process in the mammalian ovary during follicular growth. Recent reports have demonstrated the presence of pro-apoptotic and pro-autophagic proteins during oocyte elimination. The goal of this study was to identify the interactions between proteins involved in different types of programmed cell death in the same oocyte during follicular atresia. We evaluated the presence of Beclin 1 and its interaction with the pro-apoptotic proteins active caspase-3, Bax, and Bak by means of histochemical observations, ultrastructural immunodetection, and immunoprecipitation techniques in ovaries from prepubertal (28- and 33-day-old), juvenile (40-day-old), and young adult (90-day-old) rats. In this study, we identified that oocyte elimination occurred with a high quantity of pro-autophagic protein Beclin 1 and increased the presence of the pro-apoptotic proteins active caspase-3, Bax, and Bak. Conversely, the antiapoptotic protein Bcl-2 was reduced in oocytes from atretic follicles. In addition, Beclin 1 was shown to interact with active caspase-3 and Bax. Our results suggest that the increase in Beclin 1 is directly related to the rise of pro-apoptotic proteins, which could promote the apoptotic process during oocyte elimination.

    更新日期:2019-11-27
  • Transitional Hybrid Skeletal Muscle Fibers in Rat Soleus Development
    J. Histochem. Cytochem. (IF 2.37) Pub Date : 2019-09-11
    Lauren Larson; Jessica Lioy; Jordan Johnson; Scott Medler

    Skeletal muscles comprise hundreds of individual muscle fibers, with each possessing specialized contractile properties. Skeletal muscles are recognized as being highly plastic, meaning that the physiological properties of single muscle fibers can change with appropriate use. During fiber type transitions, one myosin heavy chain isoform is exchanged for another and over time the fundamental nature of the fiber adapts to become a different fiber type. Within the rat triceps surae complex, the soleus muscle starts out as a muscle comprised of a mixture type IIA and type I fibers. As neonatal rats grow and mature, the soleus undergoes a near complete transition into a muscle with close to 100% type I fibers at maturity. We used immunohistochemistry and single fiber SDS-PAGE to track the transformation of type IIA into type I fibers. We found that transitioning fibers progressively incorporate new myofibrils containing type I myosin into existing type IIA fibers. During this exchange, distinct type I-containing myofibrils are segregated among IIA myofibrils. The individual myofibrils within existing muscle fibers thus appear to represent the functional unit that is exchanged during fiber type transitions that occur as part of normal muscle development:

    更新日期:2019-11-27
  • CTLA-4 Immunohistochemistry and Quantitative Image Analysis for Profiling of Human Cancers
    J. Histochem. Cytochem. (IF 2.37) Pub Date : 2019-10-14
    Charles Brown; Farzad Sekhavati; Ruben Cardenes; Claudia Windmueller; Karma Dacosta; Jaime Rodriguez-Canales; Keith E. Steele

    Immunotherapy has proven to be an effective means to treat some cancers, as evidenced by the approval of multiple new drugs in the last decade.1,2 In particular, several immunotherapies that target the immunoregulatory pathways cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1) and its ligand (PD-L1) are now approved to treat several common forms of solid human cancers. Despite the success of CTLA-4 and PD-1/PD-L1 antibodies, however, it remains the case that such immunotherapies fail to elicit a response in many patients. Thus, there is a clear need to continue to investigate the immune response to cancer and to explore in new ways how various cellular and molecular factors might affect the immune response in different manifestations of the disease. Especially needed are ways to better predict patient response to therapy, particularly by using markers of clinical response to CTLA-4 immunotherapy.

    更新日期:2019-11-27
  • Morphological Characteristics of Neuronal Death After Experimental Subarachnoid Hemorrhage in Mice Using Double Immunoenzymatic Technique
    J. Histochem. Cytochem. (IF 2.37) Pub Date : 2019-09-17
    Fumi Nakano; Lei Liu; Fumihiro Kawakita; Hideki Kanamaru; Yoshinari Nakatsuka; Hirofumi Nishikawa; Takeshi Okada; Masato Shiba; Hidenori Suzuki

    Subarachnoid hemorrhage (SAH) is a devastating disease. Neuronal death is an important pathophysiology in the acute phase of SAH, but the histopathological features of dying neurons have been poorly studied. Using several staining methods including terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and microtubule-associated protein 2 (MAP-2) double immunolabeling, we investigated the morphological changes of nucleus and cytoskeleton in neurons and sought susceptible areas to neuronal death in filament perforation SAH mice under light microscope. TUNEL and MAP-2 double immunolabeling clearly showed morphological features of shrunken cytoplasm and sometimes curl-like fibers in dying neurons, besides nuclear abnormalities. More dying neurons were detected in the moderate SAH group than in the mild SAH group, and the temporal base cortex was the most susceptible area to neuronal death with deoxyribonucleic acid (DNA) damage among the cerebral cortices and hippocampus at 24 hr after SAH (p<0.01, ANOVA). Lesser hippocampal neuronal death was observed at 24 hr, but neuronal death was significantly increased in the CA1 region at 7 days after SAH (p<0.05, unpaired t-test). Using TUNEL and MAP-2 double immunolabeling, morphological features of not only the nucleus but also the cytoplasm in post-SAH neuronal death with DNA damage can be observed in detail under light microscope:

    更新日期:2019-11-27
  • Role of 72-kDa Heat Shock Protein in Heat-stimulated Regeneration of Injured Muscle in Rat
    J. Histochem. Cytochem. (IF 2.37) Pub Date : 2019-06-24
    Katsuya Kami; Takashi Ohira; Yasuharu Oishi; Takayuki Nakajima; Katsumasa Goto; Yoshinobu Ohira

    The regeneration of injured muscles is facilitated by intermittent heat stress. The 72-kDa heat shock protein (HSP72), the level of which is increased by heat stress, is likely involved in this effect, but the precise mechanism remains unclear. This study was conducted to investigate the localization and role(s) of HSP72 in the regenerating muscles in heat-stressed rats using immunohistochemistry. Heat stress was applied by immersion of the rat lower body into hot water (42C, 30 min, every other day) following injection of bupivacaine into the soleus muscles. After 1 week, we found that HSP72 was expressed at high levels not only in the surviving myofibers but also in the blood vessels of the regenerating muscles in heated rats. In addition, leukocytes, possibly granulocytes, expressing cluster of differentiation 43 within the blood capillaries surrounding the regenerating myofibers also highly expressed HSP72. In contrast, marked expression of HSP72 was not observed in the intact or regenerating muscles without heat stress. These results suggest that heat-stress-induced HSP72 within the myofibers, blood vessels, and circulating leukocytes may play important roles in enhancing regeneration of injured muscles by heat stress. Our findings would be useful to investigate cell-specific role(s) of HSP72 during skeletal muscle regeneration.

    更新日期:2019-11-04
  • Elevated ATF4 Expression in Odontogenic Keratocysts Epithelia: Potential Involvement in Tissue Hypoxia and Stromal M2 Macrophage Infiltration
    J. Histochem. Cytochem. (IF 2.37) Pub Date : 2019-08-19
    Wen-Qun Zhong; Zhi-Zheng Li; Hao Jiang; Yan-Ping Zou; Hai-Tao Wang; Yu Cai; Yi Zhao; Ji-Hong Zhao

    The aim of this study was to investigate the expression of the activating transcription factor 4 (ATF4) in odontogenic keratocysts (OKC), its association with hypoxia and M2-polarized macrophages infiltration, and its potential relationships with angiogenesis in OKC. The expression of ATF4, hypoxia-inducible factor 1α (HIF-1α), macrophage colony-stimulating factor (M-CSF), and receptor activator of nuclear factor κ-B ligand (RANKL) in OKC samples and normal oral mucosa (OM) was detected by immunohistochemistry. Meanwhile, microvessel density (MVD) was measured using antibody against CD31. M2-polarized macrophages were identified using double-staining for CD68+ and CD163+. The correlations of ATF4 with HIF-1α, M-CSF, and M2-polarized macrophages infiltration were determined by Spearman’s rank correlation test and hierarchical clustering. Human immortalized oral epithelial cells (HIOECs) were used in in vitro experiments. Our data showed that the expression of HIF-1α, ATF4, and M-CSF was significantly upregulated in the epithelium of OKC when compared with the OM. The expression of ATF4 was positively correlated with that of HIF-1α, M-CSF, MVD, and M2-polarized macrophages infiltration. Elevated expression of ATF4 in the epithelial lining of OKC may facilitate the M2 macrophages infiltration in response to hypoxia, leading to the development of OKC.

    更新日期:2019-11-04
  • KIF11 as a Potential Marker of Spermatogenesis Within Mouse Seminiferous Tubule Cross-sections
    J. Histochem. Cytochem. (IF 2.37) Pub Date : 2019-08-19
    Miki Hara-Yokoyama; Hidetake Kurihara; Shozo Ichinose; Hironori Matsuda; Shizuko Ichinose; Masaru Kurosawa; Norihiro Tada; Chihiro Iwahara; Kazue Terasawa; Katarzyna A. Podyma-Inoue; Koichi Furukawa; Kazuhisa Iwabuchi

    The arrangement of immature germ cells changes regularly and periodically along the axis of the seminiferous tubule, and is used to describe the progression of spermatogenesis. This description is based primarily on the changes in the acrosome and the nuclear morphology of haploid spermatids. However, such criteria cannot be applied under pathological conditions with arrested spermatid differentiation. In such settings, the changes associated with the differentiation of premeiotic germ cells must be analyzed. Here, we found that the unique bipolar motor protein, KIF11 (kinesin-5/Eg5), which functions in spindle formation during mitosis and meiosis in oocytes and early embryos, is expressed in premeiotic germ cells (spermatogonia and spermatocytes). Thus, we aimed to investigate whether KIF11 could be used to describe the progression of incomplete spermatogenesis. Interestingly, KIF11 expression was barely observed in haploid spermatids and Sertoli cells. The KIF11 staining allowed us to evaluate the progression of meiotic processes, by providing the time axis of spindle formation in both normal and spermatogenesis-arrested mutant mice. Accordingly, KIF11 has the potential to serve as an excellent marker to describe spermatogenesis, even in the absence of spermatid development.

    更新日期:2019-11-04
  • Marker Expression of Interstitial Cells in Human Skeletal Muscle: An Immunohistochemical Study
    J. Histochem. Cytochem. (IF 2.37) Pub Date : 2019-08-14
    Eva K. Hejbøl; Mohammad A. Hajjaj; Ole Nielsen; Henrik D. Schrøder

    The myogenic stem cell, the satellite cell has been intensively studied since its discovery in 1961.1 However, it becomes increasingly clear that the entire microenvironment supporting the myogenic cells must be taken into account to understand the regeneration process. It has thus been suggested that besides the satellite cell, several interstitial mesenchymal cell types contribute to regeneration by supporting myofiber formation and angiogenesis.2–4

    更新日期:2019-11-04
  • Early Alterations of Endothelial Nitric Oxide Synthase Expression Patterns in the Guinea Pig Cochlea After Noise Exposure
    J. Histochem. Cytochem. (IF 2.37) Pub Date : 2019-09-11
    Ulf R. Heinrich; Irene Schmidtmann; Regina Meuser; Benjamin P. Ernst; Desiree Wünsch; Svenja Siemer; Alena Gribko; Roland H. Stauber; Sebastian Strieth

    Constitutively expressed endothelial nitric oxide synthase (eNOS) is supposed to play a role in noise-induced nitric oxide (NO)-production. It is commonly known that intense noise exposure results in inducible NOS (iNOS) expression and increased NO-production, but knowledge about a contribution of the eNOS isoform is still lacking. Effects of noise exposure on eNOS immunolabeling were determined in male guinea pigs (n=24). For light microscopic analysis, 11 animals were exposed to 90 dB for 1 hr and 6 animals were used as controls. After exposure, eNOS immunostaining was performed on paraffin sections, and the staining intensities were quantified for 4 cochlear regions. For electron microscopic analysis, 2 animals were exposed for 2 hr to 90 dB and 5 animals were used as controls. The intensity of eNOS immunolabeling was found to be already comprehensively increased 1 hr after noise exposure to 90 dB. At the ultrastructural level, a clear increase in eNOS immunolabeling was found in microtubules-rich areas of cochlear cuticular structures. Hence, our findings indicate that the reticular lamina forming the endolymph–perilymph barrier at the apical side of the organ of Corti is involved in a fast intrinsic otoprotective mechanism of the cochlea.

    更新日期:2019-11-04
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