Toluidine blue staining of murine mast cells and quantitation by a novel, automated image analysis method using whole slide skin images.

Mast cells are immune cells of myeloid lineage, characterized by the presence of cytoplasmic granules. These cells play a significant role in multiple pathologies, such as urticaria and type 1 hypersensitivity reactions. Mast cells in tissue sections can be demonstrated with metachromatic stains, such as toluidine blue. Metachromatic staining of mast cells is influenced by the type of mast cell, pH of the staining solution and fixative employed. The objective of this study is to identify a simple, consistent, and reproducible toluidine blue staining method to quantify the mast cells in whole slide skin images using automated image analysis. Skin sections from naive mice and atopic dermatitis mice were stained with toluidine blue methods by Churukian-Schenk and Luna. Luna's toluidine blue staining method without the eosin counterstaining provided optimal staining of mast cells with a greater contrast to the background. The Churukian-Schenk toluidine blue method resulted in slight background staining which confounded the accurate detection and quantification of mast cells in mouse skin sections using an automated image analysis algorithm. Using the Luna toluidine blue stain, a 5-fold increase in the total number of mast cells was observed in atopic dermatitis skin samples as compared to naive mice. In summary, a simple, conventional, and reproducible toluidine blue method was identified to quantify the mast cells in mouse skin sections using an automated image analysis algorithm.

Differential expression of mast cell granules in samples of metastatic and non-metastatic colorectal cancer in patients.

Various cell types participate in the tumor process, in which the mast cells have been described; however, the role they play in colorectal adenocarcinoma has not yet been fully understood. Therefore, the present work aimed to compare employing histochemistry and immunohistochemistry, the number of mast cells and the content of some cytoplasmic granules in moderately differentiated non-metastatic and metastatic colorectal adenocarcinoma, analyzing tissue samples from patients. Histochemical techniques with Toluidine Blue (TBO), Periodic Schiff Acid (PAS), Alcian Blue/Periodic Acid-Schiff (PAB) and Alcian Blue/Safranin (ABS); as well as immunohistochemical reactions with anti-antibodies anti-Tryptase and anti-Chymase were applied to quantify total mast cells and content of some cytoplasmic granules. Statistical analysis was performed using SPSS V22.0 software (p ≤ 0.05). The degree of positivity of the reaction and degranulation of mast cells was reported in percentages. In our results, we observed that there are differences in the quantity and histochemical composition of the granules of mast cells (metastatic group PAS and ABS comparing the TBO reaction), as well as in the immunohistochemical composition between Tryptase and Chymase and the number of degranulated cells in both study groups (74 % degranulated mast cells in the metastatic group, 66 % integrate mast cells in the non-metastatic group). Therefore, we consider that the differences may be some of the probable factors that lead to metastasis of colorectal adenocarcinoma.

Sealing capability of implant-abutment junction under cyclic loading: a toluidine blue in vitro study.

PURPOSE: The aim of the present in vitro study was to evaluate the leakage observed in external hexagon (EH) and cone Morse (CM) tapered implant-abutment connections, using toluidine blue. METHODS: A total of 60 implants, 30 with a screw-retained EH abutment and 30 with a CM taper internal connection, were used. Toluidine blue was placed into the deepest portion of the internal compartment of the 2 different implant systems, and cyclic loading was applied for each group as follows: 10 samples underwent 1 × 10(6) loading cycles, 10 samples underwent 3 × 10(6) cyclic loading and the least 10 samples underwent 6 × 10(6) cyclic loading. RESULTS: No significant differences between the EH and CM groups were detected when the lowest loading cycles were applied (p = 0.2624), while differences were found when the samples were loaded with 3 x 10(6) and 6 x 10(6) cycles (p = 0.00124), with significantly lower toluidine leakage in CM group. CONCLUSIONS: In conclusion, the results of the present in vitro study demonstrated that flow of the toluidine blue to the external portion of the implant-abutment assembly occurred in both types of implant-abutment connections, with very different percentages. Indeed, the CM taper internal connection seems to be more resistant to the leakage of dyes when compared with EH connections.

Quantitative surface-enhanced Raman measurements with embedded internal reference.

Analytical applications of SERS are often more associated with qualitative than quantitative analysis, because of the difficulty in obtaining quantitative SERS results. In this paper we introduce a new strategy to quantitatively measure the SERS signals of analytes based on Au-core/Ag-shell nanoparticles with embedded 4-aminothiophenol as the internal reference. Successful detections of two analytes, Toluidine Blue O in aqueous solution (detection limit of 0.1 µM) and melamine in milk (detection limit of ~5 µM), are demonstrated. The improvement in the linear fitting illustrates that the use of internal reference significantly improves the accuracy of the quantitative SERS measurements. The successful detection of melamine in milk illustrates the versatility of this detection scheme for a wide variety of analytes.

Analysis of RNA by analytical polyacrylamide gel electrophoresis.

Polyacrylamide gel electrophoresis (PAGE) is a powerful tool for analyzing RNA samples. Denaturing PAGE provides information on the sample composition and structural integrity of the individual RNA species. Nondenaturing gel electrophoresis allows separation of the conformers and alternatively folded RNA species. It also can be used to resolve RNA protein complexes and to detect RNA complex formation by analyzing changes in the electrophoretic mobility of the RNA. RNA can be visualized within gels by different methods depending on the nature of the detection reagent. RNA molecules can be stained with various dyes, including toluidine blue, SYBR green, and ethidium bromide. Radioactively labeled RNA molecules are visualized by autoradiography, and fluorescently labeled RNA molecules can be observed with a fluorescence scanner. Generally, gels between 0.4 and 1.5mm thick are used for analytical PAGE. Gels thinner than 1mm are fragile and thus usually are not stained but rather are used for radiolabeled RNA. The gels are dried and the radiolabeled RNA is visualized by autoradiography.

Techniques for the study of apoptosis in bone.

There has been great interest in the identifying the mechanisms by which apoptosis is regulated in bone over recent years and in the biological role that this process plays in bone metabolism and bone disease. Here, we describe several methods for the detection of apoptosis in bone sections and in bone cell cultures.

Epidermal expression of an Elovl4 transgene rescues neonatal lethality of homozygous Stargardt disease-3 mice.

Elongase of very long chain fatty acids-4 (ELOVL4) is the only mammalian enzyme known to synthesize C28-C36 fatty acids. In humans, ELOVL4 mutations cause Stargardt disease-3 (STGD3), a juvenile dominant macular degeneration. Heterozygous Stgd3 mice that carry a pathogenic mutation in the mouse Elovl4 gene demonstrate reduced levels of retinal C28-C36 acyl phosphatidylcholines (PC) and epidermal C28-C36 acylceramides. Homozygous Stgd3 mice die shortly after birth with signs of disrupted skin barrier function. In this study, we report generation of transgenic (Tg) mice with targeted Elovl4 expression driven by an epidermal-specific involucrin promoter. In homozygous Stgd3 mice, this transgene reinstates both epidermal Elovl4 expression and synthesis of two missing epidermal lipid groups: C28-C36 acylceramides and (O-linoleoyl)-omega-hydroxy C28-C36 fatty acids. Transgene expression also restores skin barrier function and rescues the neonatal lethality of homozygous Stgd3 mice. These studies establish the critical requirement for epidermal C28-C36 fatty acid synthesis for animal viability. In addition to the skin, Elovl4 is also expressed in other tissues, including the retina, brain, and testes. Thus, these mice will facilitate future studies to define the roles of C28-C36 fatty acids in the Elovl4-expressing tissues.

An easy method to highlight chief cells in gastric biopsies.

BACKGROUND: Hematoxylin and eosin (H&E) is not an optimal stain to discriminate chief cells from parietal cells in gastric biopsies MATERIALS AND METHODS: Fifteen sets of biopsies from the gastric corpus were consecutively stained with H&E and toluidine blue stains; chief cells stained deep blue with toluidine blue, thus contrasting with lightly-stained parietal cells. In well-oriented sections, the continuity of the chief cell zone was studied at × 4 magnification and its thickness in one field at × 10 magnification. RESULTS: Toluidine blue stained fundic sections that exhibited normal mucosa or chronic gastritis without glandular atrophy displayed a distinct deep-blue chief cell zone, intercalated between the lightly-stained parietal cells on top and the muscularis mucosae underneath (Group A). In chronic gastritis with focal glandular atrophy or focal intestinal metaplasia, at least one focally fragmented toluidine blue chief cell zone was seen (Group B). In one case with severe autoimmune gastritis and in a case with extensive intestinal metaplasia, an absence of toluidine blue chief cells zone was recorded in the entire section (Group C). CONCLUSION: This quick and easy staining method made it possible to group sections from the gastric corpus into those with a continuous chief cell zone, with fragmented or with an absent chief cell zone, modalities that seem to correlate with different stages of fundic mucosal inflammation. These preliminary results should be validated in a larger cohort of gastric biopsies from patients with various diseases affecting the corpus mucosa.

Análise computacional da compactação da cromatina de espermatozoides de galo / Computational analysis of chromatin condensation of rooster spermatozoa

Testaram-se variantes metodológicas utilizando azul de toluidina (AT), até se estabelecer um protocolo confiável para a avaliação computacional da compactação da cromatina em espermatozoides de galo. Para tal, foram utilizados sêmen de 10 galos com 35 semanas de idade e sêmen de 10 galos com 60 semanas de idade. O melhor método foi o de hidrólise com ácido clorídrico 1N por 10 minutos, coloração em cubeta com AT 0,025 por cento, pH 4,0, por 20 minutos, desidratação em álcool, diafanização em xilol e montagem com bálsamo do Canadá. Todas as amostras de sêmen foram submetidas a este protocolo e posteriormente avaliadas por análise de imagem computacional, em que foram feitas mensurações da área, comprimento, largura, perímetro, homogeneidade da compactação da cromatina dentro de cada cabeça e intensidade de compactação da cromatina. Os espermatozoides de galos velhos apresentaram mais alterações na cromatina que os de galos jovens. Os galos jovens apresentaram cabeça dos espermatozoides maior que os galos mais velhos. A análise computacional da compactação da cromatina mostrou-se um método menos subjetivo e mais preciso que a avaliação visual das cabeças dos espermatozoides.

The methodological variants using toluidina blue (AT) to establish a trustworthy protocol for the computational analysis of chromatin condensation of rooster spermatozoa were studied. Twenty semen samples were used: ten from 35-week-old roosters and ten from 60-week-old roosters. Different methods of denaturation and staining were tested. The best method was hydrolysis with 1N HCl for 10 minutes, staining in bucket with 0.025 percent AT, pH 4.0, for 20 minutes, dehydration in alcohol, clearing in xylol, and mounted with Canada balsam. All the semen samples were submitted to this protocol and later evaluated by computational image analysis. Area, length, width, perimeter, and chromatin compaction homogeneity of head spermatozoa were measured. The sperm of older roosters presented more chromatin changes than the ones of younger ones. The spermatozoa of younger roosters presented bigger heads than the ones of older roosters. The computational analysis of chromatin compaction showed to be less subjective and more precise than the visual evaluation for identification of chromatin alterations of rooster spermatozoa.

Multiplex binding modes of toluidine blue with calf thymus DNA and conformational transition of DNA revealed by spectroscopic studies.

It is noteworthy to understand the details of interactions between antitumor drugs and DNA because the binding modes and affinities affect their antitumor activities. Here, The interaction of toluidine blue (TB), a potential antitumor drug for photodynamic therapy of tumor, with calf thymus DNA (ctDNA) was explored by UV-vis, fluorescence, circular dichroism (CD) spectroscopy, UV-melting method and surface-enhance Raman spectroscopy (SERS). The experimental results suggest that TB could bind to ctDNA via both electrostatic interaction and partial intercalation. The fluorescence quenching of TB by ctDNA was static and due to electron transfer from bases to the excited singlet state of TB. At low [TB]/[DNA] ratio, TB mainly partially intercalated into ctDNA resulting in the slight increase of base stacking degree; at high [TB]/[DNA] ratio, excessive TB externally stacked along the helix surface via coupling with partially intercalated ones, thereby inducing B-A transition of ctDNA. The conformational transition of DNA was confirmed by the obvious improvement of the thermal stability of ctDNA. The SERS spectra suggest that TB could partially intercalate into DNA basepairs with its ring C(1)NC(1') side buried.

[Determination of nucleic acids using toluidine blue as a fluorescence probe].

A new fluorimetric method has been developed for the determination of deoxyribonucleic acid (DNA) with toluidine blue (TB) as a fluorescence probe. It is based on the fluorescence quenching of toluidine blue in the presence of DNA. In Tris-HCl buffer solution (pH 8.5), the calibration graph was linear over the range 0.10-6.00 microg x mL(-1) for ctDNA, and the detection limit was 27 ng x mL(-1). The result of the determination of DNA of camphor tree's leaf by this method was satisfactory.

Revisión de la aplicación clínica de los colorantes en cromoendoscopia digestiva y su formulación magistral / review of the clinical application of dyes in gastrointestinal chromoendoscopy, and their magistral formulation

Objetivo: El objetivo del presente trabajo es revisar la formulación magistral de los colorantes empleados y su efectividad clínica. Método: Se realizó una búsqueda bibliográfica en Medline, Cochrane Library y Micromedex, utilizando el término cromoendoscopia y repitiendo la búsqueda con los colorantes localizados asociados al término endoscopia. Se revisaron también diversas monografías, revistas científicas y las citas de los trabajos seleccionados. Los trabajos recuperados se clasificaron en función de su metodología. Resultados: Se localizaron 96 referencias, recuperándose el artículo original sólo en 57 de ellas correspondientes a 13 ensayos clínicos, 21 series de casos y 11 revisiones. Se encontraron referencias para 7 colorantes. Se describen las principales indicaciones, las evidencias de efectividad, la forma de administración y la fórmula magistral para cada uno de los colorantes. Conclusiones: El número de trabajos es elevado aunque la accesibilidad a los mismos es limitada. La evidencia es escasa en conjunto aunque en determinados casos como el azul de metileno en esófago de Barrett, el lugol en la detección de carcinomas esofágicos y el índigo carmín en la diferenciación de pólipos hiperplásicosa nivel colónico es alta. La formulación magistral es bastante simple aunque está poco desarrollada

Objective: To review the drug compounding of dyesemployed in chromoendoscopy, and their clinical effectiveness. Method: A literature search in Medline, Cochrane Library, and Micromedex was carried out with the term chromoendoscopy as a keyword, and the search was then repeated for each dye found in association with the term endoscopy. A number of monographs, scientific journals, and references quoted in selected papers were also reviewed. Papers collected were then classifiedaccording to their methodology. Results: Ninety-six references were found, their original article being recovered for only 57 of these – 13 clinical trials, 21case series, and 11 reviews. References were found for 7 dyes. Main indications, evidence of effectiveness, administration route, and drug formulation are described for each dye. Conclusions: The number of papers involved is high, but their accessibility is limited. Evidence is overall scarce, but high in cases such as methylene blue for Barrett's esophagus, lugol in the detection of esophageal carcinoma, and indigo carmine for colonic hyperplastic polyp differentiation. Drug compounding is rather simple, but scarcely developed

[Toluidine blue in monitoring of recurrences and second primary tumors in patients treated for oral cavity and pharynx cancer]. / Zastosowanie blqkitu toluidyny do monitorowania wznów i drugich pierwotnych ognisk nowotworowych w jamie ustnej i gardle u chorych leczonych z powodu raka jezyka i migdalka podniebiennego.

UNLABELLED: Assesment of recurrences in the oral cavity and pharynx is a serious diagnostic challenge particularly in patient after surgery and irradiation. Observed visible local lesions like oedema, formation of postoperative scars and pain made the diagnostic procedure difficult. Sometimes it's impossible to distinguish recurrences and inflammatory reaction. The aim of this research was to evaluate the usefulness of toluidyne blue in monitoring of recurrences and second primary tumors in the patients with oral and pharyngeal cancer. It was perform by comparison of the three methods: physical examination, endoscopic ultrasound probe and toluidine blue staining in detection of early malignancy. A group of 80 patients with oral and pharyngeal squamous cell cancer was treated in the years between 2000-2003 in the Department of Otolaryngology in Poznan. In 7 positive tested cases toluidine blue staining was confirmed in histological examination. 3 cases were false-positive staining (patients after reconstructive with infrahyoid and pectoral major flap). CONCLUSIONS: (1) toluidine blue staining is cheap effective diagnostic procedure in monitoring recurrences in patients after surgery and irradiation; (2) procedure is limited in patients after reconstructive flap surgery; (3) ultrasound endoscopy is of value in assesment of advanced tumors of oral cavity and pharynx, but it's limited in flat and superficial mucosal infiltrations.

Morphological and immunocytochemical investigations on mast cells in porcine ureter.

Morphological, morphometric, histochemical and immunocytochemical investigations on mast cells, located in the wall of ureter of 8 months aged pigs were performed. Mast cells were found in all three layers of ureteral wall, but their distribution was irregular and the number unequal. It was established that alcian blue (AB)-positive mast cells were significantly more than toluidine blue (TB)-positive mast cells. A statistically significant smaller number of both AB and TB-stained mast cells were observed in the tunica mucosa. The largest number of mast cells was found in the tunica muscularis. In the adventitia, mast cells were higher in number in the main connective tissue than in the connective tissue near the blood vessels. Mast cells stained with TB showed variably expressed gamma-metachromasia, which was best visible in those situated in the lamina propria of the mucosa. The prevailing parts of mast cells, however, were AB-positive after AB-safranin staining. This was mostly found in mast cells of the tunica muscularis and in mast cells of perivascular location in the tunica adventitia. Immunocytochemically, mast cells were found to be positive for histamine and vasoactive intestinal polypeptide in the muscle coat, and to histamine in the adventitia, as well. On the basis of obtained results it was presumed that the mast cells in porcine ureter most probably took part not only in keeping of local homeostasis, but played also an important role of mobility of smooth muscle cells in the middle layer of ureter on one hand, and, on the other, in the adventitial blood vessels.

Destructive effects of murine arthritogenic antibodies to type II collagen on cartilage explants in vitro.

Certain monoclonal antibodies (mAbs) to type II collagen (CII) induce arthritis in vivo after passive transfer and have adverse effects on chondrocyte cultures and inhibit self assembly of collagen fibrils in vitro. We have examined whether such mAbs have detrimental effects on pre-existing cartilage. Bovine cartilage explants were cultured over 21 days in the presence of two arthritogenic mAbs to CII (CIIC1 or M2139), a non-arthritogenic mAb to CII (CIIF4) or a control mAb (GAD6). Penetration of cartilage by mAb was determined by immunofluorescence on frozen sections and correlated with changes to the extracellular matrix and chondrocytes by morphometric analysis of sections stained with toluidine blue. The effects of mAbs on matrix components were examined by Fourier transform infrared microspectroscopy (FTIRM). A possible role of Fc-binding was investigated using F(ab)2 from CIIC1. All three mAbs to CII penetrated the cartilage explants and CIIC1 and M2139, but not CIIF4, had adverse effects that included proteoglycan loss correlating with mAb penetration, the later development in cultures of an abnormal superficial cellular layer, and an increased proportion of empty chondrons. FTIRM showed depletion and denaturation of CII at the explant surface in the presence of CIIC1 or M2139, which paralleled proteoglycan loss. The effects of F(ab)2 were greater than those of intact CIIC1. Our results indicate that mAbs to CII can adversely affect preformed cartilage, and that the specific epitope on CII recognised by the mAb determines both arthritogenicity in vivo and adverse effects in vitro. We conclude that antibodies to CII can have pathogenic effects that are independent of inflammatory mediators or Fc-binding.

Improved demonstration of mast cells using alcian blue tetrakis (methylpyridium) chloride.

Many batches of alcian blue dye are incompletely soluble at the low pH used for demonstrating mast cells. An improved technique using alcian blue tetrakis (methylpyridium) chloride (alcian blue pyridine variant) is described here. It produces stronger mast cell staining than other alcian blue stains tested.

Aplicação clínica de corantes como auxílio diagnóstico em Estomatologia / Clinical application of colorings as aid diagnosis in oral pathology

Os autores apresentam tipos de corantes que têm sido utilizados na prática de evidenciação de lesões bucais, abordando sua indicação e técnica de aplicação. Concluem pela não indicação de seu uso como rotina e deixam a indicação para trabalho a longo prazo, em que seria possível uma avaliação sobre resultados concretos

Chromatographic, mass spectral, and visible light absorption characteristics of toluidine blue O and related dyes..

Contrary to earlier literature reports, the impurities in toluidine blue O were shown by column chromatography, TLC, and mass spectrometry to be N-methyl homologs of 2-methylthionine rather than N-methyl homologs of thionine. Small amounts of 2-methyl-3-amino-7-methylaminophenothiazine and 2-methyl-3,7-diaminophenothiazine were identified in commercial samples of toluidine blue O. However, sample handling and a warm alkaline environment can cause rapid demethylation of the dye.