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Concept: Staining

171

Spinal ligaments, such as the ligamentum flavum (LF), are prone to degeneration and iatrogenic injury that can lead to back pain and nerve dysfunction. Repair and regeneration strategies for these tissues are lacking, perhaps due to limited understanding of spinal ligament formation, the elaboration of its elastic fibers, maturation and homeostasis. Using immunohistochemistry and histology, we investigated murine LF elastogenesis and tissue formation from embryonic to mature postnatal stages. We characterized the spatiotemporal distribution of the key elastogenic proteins tropoelastin, fibrillin-1, fibulin-4 and lysyl oxidase. We found that elastogenesis begins in utero with the microfibril constituent fibrillin-1 staining intensely just before birth. Elastic fibers were first detected histologically at postnatal day (P) 7, the earliest stage at which tropoelastin and fibulin-4 stained intensely. From P7 to P28, elastic fibers grew in diameter and became straighter along the axis. The growth of elastic fibers coincided with intense staining of tropoelastin and fibulin-4 staining, possibly supporting a chaperone role for fibulin-4. These expression patterns correlated with reported skeletal and behavioral changes during murine development. This immunohistochemical characterization of elastogenesis of the LF will be useful for future studies investigating mechanisms for elastogenesis and developing new strategies for treatment or regeneration of spinal ligaments and other highly elastic tissues.

Concepts: Liver, Anatomical pathology, Histology, Osteoarthritis, Immunohistochemistry, Staining, Ligament, Microtome

171

Glycerol monolaurate (GML) is an antimicrobial agent that has potent activity against gram-positive bacteria. This study examines GML antibacterial activity in comparison to lauric acid, in broth cultures compared to biofilm cultures, and against a wide range of gram-positive, gram-negative, and non-gram staining bacteria.

Concepts: Bacteria, Microbiology, Staining, Gram-negative bacteria, Tea tree oil, Gram staining, Gram-positive bacteria, Bacteriology

140

Woodpeckers experience forces up to 1200-1400 g while pecking. It is assumed due to evolutionary adaptations, the woodpecker is immune to brain injury. This assumption has led to the use of the woodpecker as a model in the development of sports safety equipment such as football helmets. However, it is unknown at this time if the woodpecker brain develops neuro-trauma in relation to the high g-forces experienced during pecking. The brains of 10 ethanol preserved woodpeckers and 5 ethanol preserved red-winged black bird experimental controls were examined using Gallyas silver stain and anti-phospho-tau. The results demonstrated perivascular and white matter tract silver-positive deposits in eight out of the 10 woodpecker brains. The tau positive accumulations were seen in white matter tracts in 2 of the 3 woodpeckers examined. No staining was identified in control birds. The negative staining of controls birds contrasted with the diffuse positive staining woodpecker sections suggest the possibility that pecking may induce the accumulation of tau in the woodpecker brain. Further research is needed to better understand the relationship.

Concepts: Traumatic brain injury, Bird, White matter, Staining, Silver stain, Negative stain, Woodpecker, Phosphotungstic acid

73

Obtaining high-resolution information from a complex system, while maintaining the global perspective needed to understand system function, represents a key challenge in biology. Here we address this challenge with a method (termed CLARITY) for the transformation of intact tissue into a nanoporous hydrogel-hybridized form (crosslinked to a three-dimensional network of hydrophilic polymers) that is fully assembled but optically transparent and macromolecule-permeable. Using mouse brains, we show intact-tissue imaging of long-range projections, local circuit wiring, cellular relationships, subcellular structures, protein complexes, nucleic acids and neurotransmitters. CLARITY also enables intact-tissue in situ hybridization, immunohistochemistry with multiple rounds of staining and de-staining in non-sectioned tissue, and antibody labelling throughout the intact adult mouse brain. Finally, we show that CLARITY enables fine structural analysis of clinical samples, including non-sectioned human tissue from a neuropsychiatric-disease setting, establishing a path for the transmutation of human tissue into a stable, intact and accessible form suitable for probing structural and molecular underpinnings of physiological function and disease.

Concepts: DNA, Molecular biology, Blood, Biology, Histology, System, Immunohistochemistry, Staining

29

The aims of this study were to determine the expression of Ki-67 in type I and type II endometrial adenocarcinomas as well as normal endometrium in imprint smears and to correlate the results with clinicopathologic parameters of primary untreated endometrial cancer patients. During a 29-month period, 255 patients were evaluated with entometrial imprint cytology. Endometrial samples freshly resected from women who underwent total abdominal hysterectomy were studied. One hundred twenty-six patients had endometrial carcinoma and 129 cases were diagnosed as normal endometrium. The expression of Ki-67 was assessed by immunocytochemistry. Positive staining was correlated with increased stage, grade and lymph node metastases. High expression was more frequent in type II than type I endometrial adenocarcinoma and high-grade endometrial carcinoma had higher proportions of Ki-67 positive immunostaining compared with low-grade carcinoma. Proliferative endometrium showed high Ki-67 expression level, even higher than those of grade 1 and type I. On the other hand, secretory endometrium Ki-67 positive cells were markedly diminished and even disappeared. Completely negative staining was found to be related to atrophic endometrium. Immunocytochemical findings from Ki-67 stain, in addition to cytomorphologic features, appeared to be useful for the diagnosis of endometrial carcinoma in endometrial cytology with imprint smears. High Ki-67 expression correlates with morphologic features of aggressiveness and the expression pattern of Ki-67 correspond to the expected cyclic/atrophic pattern in normal endometrium. Diagn. Cytopathol. 2013. © 2011 Wiley Periodicals, Inc.

Concepts: Cancer, Lung cancer, Menopause, Estrogen, Endometrium, Immunohistochemistry, Endometrial cancer, Staining

28

Distinguishing malignant mesotheliomas from benign mesothelial proliferations on hematoxylin and eosin-stained sections can be extremely challenging. Various immunohistochemical stains have been suggested to help in making this distinction, but all are controversial. Recently, IMP3 (insulin-like growth factor II mRNA binding protein 3) and GLUT-1 (glucose transporter protein 1) have been proposed as immunohistochemical markers that are positive in mesotheliomas but not in benign proliferations. We evaluated the performance of these markers on a tissue microarray containing 30 malignant mesotheliomas and 48 benign thoracic or abdominal mesothelial proliferations. IMP3 was positive in 53% of malignant and 27% of benign cases (P=0.03), whereas GLUT-1 was positive in 60% of malignant and 13% of benign cases (P=0.0003). Forty-three percent of malignant cases, but only 4% of benign cases, were positive for both IMP3 and GLUT-1 (P=0.00003). We conclude that, statistically, both IMP3 and GLUT-1 are more frequently positive in malignant compared with benign mesothelial processes; however, the frequency of positive staining in benign cases is too high to allow their diagnostic use as single stains. The combination of both markers may be of greater diagnostic value, but this hypothesis should be confirmed in further studies.

Concepts: Antibody, Protein, Glucose, Histology, Immunohistochemistry, Glucose transporter, Staining, GLUT1

28

The TiO(2) nanowire (TiO(2) Nw) was successfully prepared via hydrothermal method through TiO(2) nanoparticle (TiO(2) Np). TiO(2) Np doped silver and TiO(2) Nw doped silver were prepared via photo-reducing Ag(+) ions to Ag metal on the TiO(2) Np or TiO(2) Nw surfaces. The prepared nanomaterials were evaluated using X-ray (XRD) diffraction pattern, scanning electron microscopy (SEM), and transmission electron microscopy (TEM). Bleached untreated cotton fabric and PVP treated cotton fabrics were coated with the synthesized nanomaterials using pad-dry-cure method. Photocatalytic activity of untreated and coated cotton fabrics with TiO(2) nanomaterials was investigated through the fabric self cleaning of MB dye stains. Also, the PVP finished cotton fabric modified by nanomaterials demonstrated antimicrobial activity against Gram positive bacteria, Gram negative bacteria and fungi. The mechanical properties of coated cotton fabric (tear strength, surface roughness, tensile strength and elongation at break) were examined.

Concepts: Electron, Microbiology, Transmission electron microscopy, Scanning electron microscope, Staining, Gram-negative bacteria, Textile, Gram-positive bacteria

28

Immunohistochemical staining for DNA mismatch repair proteins may be affected by various biological and technical factors. Staining variations that could potentially lead to erroneous interpretations have been recognized. A recently recognized staining variation is the significant reduction of staining for MSH6 in some colorectal carcinomas. The frequency and specific characteristics of this aberrant MSH6 staining pattern, however, have not been well analyzed. In this study of 420 colorectal carcinoma samples obtained from patients fulfilling the Revised Bethesda Guidelines, we detected 9 tumors (2%) showing extremely limited staining for MSH6 with positive staining present in <5% of the tumor cells. Our analyses showed that these tumors belonged to two distinct categories: (1) MLH1 and/or PMS2 protein-deficient carcinomas (n=5, including 1 with a pathogenic mutation in PMS2); and (2) MLH1, PMS2 and MSH2 normal but with chemotherapy or chemoradiation therapy before surgery (n=4). To test our hypothesis that somatic mutation in the coding region microsatellite of the MSH6 gene might be a potential underlying mechanism for such limited MSH6 staining, we evaluated frameshift mutation in a (C)(8) tract in exon 5 of the MSH6 gene in seven tumors that had sufficient DNA for analysis, and detected mutation in four; all four tumors belonged to the MLH1/PMS2-deficient group. In conclusion, our data outline the main scenarios where significant reduction of MSH6 staining is more likely to occur in colorectal carcinoma, and suggest that somatic mutations of the coding region microsatellites of the MSH6 gene is an underlying mechanism for this staining phenomenon in MLH1/PMS2-deficient carcinomas.

Concepts: DNA, Genetics, Cancer, Mutation, DNA repair, Anatomical pathology, Frameshift mutation, Staining

28

Tissue microarrays were originally developed to enable alignment of multiple tissue cores in a single paraffin block and to enable high-throughput laboratory analysis. However, a major drawback is the loss of tissue cores during slide preparation, especially when sectioning the tissue block. Tissue cylinders directly aligned in the metal box without preheating tend to detach from the surrounding paraffin, which results in incomplete or folded tissue sections. The proposed solution is preheating all tissue cylinders on a hot plate to facilitate fusion between the paraffin within the core and the paraffin surrounding the core. In this study, 6 tissue microarray blocks were constructed from 528 tissue cores extracted from various formalin-fixed, paraffin-embedded human tissue samples. The tissue cores in the arrays revealed good homogenization with the surrounding paraffin wax, and the tissue sections were obtained intact. Both hematoxylin-eosin and immunohistochemical staining confirmed satisfactory results. This simple and economical method is easily performed in the laboratory without expensive instrumentation.

Concepts: Histology, Immunohistochemistry, Paraffin, Tissue, Staining, Microarray, Array, Tissue microarray

28

Those investigators who study the morphology of Legionella and Legionella-infected cells have greatly benefited from the superior resolution afforded by electron microscopy (EM). It can also be said with confidence that EM will continue to reveal as yet to be discovered features of this fascinating intracellular pathogen. In this chapter we detail our practical experience in the application of three transmission electron microscopy (TEM) techniques to the study of Legionella: conventional ultrastructural analysis, immuno-gold labeling, and negative staining. Each of these techniques has particular, well-defined applications, which are discussed in the context of our in-house developed methods. We invite researchers to try the methods given here in the study of Legionella, and adopt TEM as part of their research tools arsenal.

Concepts: Electron, Biology, Microscope, Transmission electron microscopy, Scanning electron microscope, Staining, Electron microscopy, Negative stain