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2.
Forensic Sci Int ; 307: 110114, 2020 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-31901461

RESUMEN

Seminal evidence obtained from a sexual crime scene provides clues for solving a case through forensic analysis. However, most evidence collected from sexual crime scenes is a mixture of sperm cells and vaginal discharge. Therefore, separating the sperm cells from the seminal evidence is very important. In this study, we developed a separation method for effectively separating sperm cells using differential extraction with commercially available sperm staining reagents such as hematoxylin and nigrosin. Hematoxylin (0.03 % v/v) effectively stained the sperm cells in ATL and TNE lysis buffer, while nigrosin did not. The loss of sperm cells during washing of the specimen was minimized using the differential extraction method. Subsequently, genomic DNA was extracted from the hematoxylin-stained sperm cells and subjected to short tandem repeat genotyping. We observed no interference from hematoxylin. These results indicate that hematoxylin can be used to stain sperm cells and thus facilitate subsequent genetic identification.


Asunto(s)
Separación Celular , Hematoxilina , Delitos Sexuales , Espermatozoides/química , Espermatozoides/citología , Compuestos de Anilina , ADN/aislamiento & purificación , Dermatoglifia del ADN , Electroforesis Capilar , Femenino , Genética Forense , Técnicas de Genotipaje , Humanos , Indicadores y Reactivos , Masculino , Repeticiones de Microsatélite , Reacción en Cadena de la Polimerasa , Coloración y Etiquetado
3.
Chemistry ; 26(2): 384-389, 2020 Jan 07.
Artículo en Inglés | MEDLINE | ID: mdl-31550056

RESUMEN

Phosphatidylinositol (PI) is the biosynthetic precursor for seven phosphoinositides, important signaling lipids in cells. A membrane-permeant caged PI derivative featuring a photo-removable coumarinyl group masking the negative charge of the phosphate, as well as two enzymatically removable butyrate esters for increased lipophilicity and for preventing phosphate migration, were synthesized. Rapid cell entry and cellular labeling in fixed cells was demonstrated by a photo-cross-linkable diazirine followed by attachment of a fluorophore through click chemistry. Using this technique, we found that the multifunctional caged PI derivative resided predominantly at internal membranes but rapidly changed to the plasma membrane after uncaging. Accordingly, a preliminary proteomic analysis of the lipid-protein conjugates revealed that the two major PI transport proteins PITPα and ß were prime targets of the photo-cross-linked PI derivative.


Asunto(s)
Fosfatidilinositoles/química , Coloración y Etiquetado/métodos , Membrana Celular/metabolismo , Química Clic , Células HeLa , Humanos , Microscopía Fluorescente , Fosfatidilinositoles/síntesis química , Fosfatidilinositoles/metabolismo
4.
Indian J Ophthalmol ; 68(1): 71-72, 2020 01.
Artículo en Inglés | MEDLINE | ID: mdl-31856471
5.
Biosci Biotechnol Biochem ; 84(1): 154-158, 2020 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-31794328

RESUMEN

Malectin is a maltose-binding endoplasmic reticulum protein conserved in animals. In Arabidopsis thaliana, we identified four genes that encode malectin-like domain (MLD)- and leucine-rich repeat (LRR)-containing proteins (AtMLLRs): two were receptor-like proteins (AtMLLR1 and 2) and the other two were extracellular proteins (AtMLLR3 and 4). The promoter:G3GFP+promoter:GUS assay indicated the organ- and cell-specific expression of the AtMLLR2 and AtMLLR3 genes.Abbreviations: Cmr: chloramphenicol-resistance marker; G3GFP: G3 green fluorescent protein; GUS: ß-glucuronidase; KD: kinase domain; LRR: leucine-rich repeat; MLD: malectin-like domain; RLK: receptor-like kinase; SP: signal peptide; TMD: transmembrane domain; Tnos: nopaline synthase terminator.


Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Expresión Génica , Lectinas/genética , Proteínas de la Membrana/genética , Proteínas/genética , Retículo Endoplásmico/metabolismo , Glucuronidasa/química , Proteínas Fluorescentes Verdes/química , Leucina/genética , Microscopía Fluorescente , Filogenia , Plantas Modificadas Genéticamente , Dominios Proteicos/genética , Coloración y Etiquetado
6.
Arch Virol ; 165(2): 367-375, 2020 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-31845151

RESUMEN

The genus Tobravirus comprises three species: Tobacco rattle virus, Pea early-browning virus and Pepper ringspot virus. The genomes of tobraviruses consist of two positive-sense single-stranded RNA segments (RNA1 and RNA2). Infectious clones of TRV are extensively used as virus-induced gene-silencing (VIGS) vectors for studies of virus-host interactions and functions of plant genes. Complete infectious clones of pepper ringspot virus (PepRSV), the only tobravirus present in Brazil, however, have not yet been reported. Infectious clones will help to identify unique features of PepRSV RNA2 and provide another option for development of VIGS vectors. We constructed infectious clones based on two PepRSV isolates, CAM (RNA1 and RNA2) and LAV (RNA2). The cDNA constructs for both homologous (RNA1 and RNA2 of the CAM isolate) and heterologous (RNA1/CAM and RNA2/LAV) combinations were infectious in Nicotiana benthamiana plants. VIGS vector constructs with green fluorescent protein or phytoene desaturase genes inserted in RNA2 silenced the target genes. The systemic translocation of the PepRSV RNA1 construct alone (nonmultiple infection) was also confirmed in an N. benthamiana plant. These results are similar to those reported for tobacco rattle virus.


Asunto(s)
Vectores Genéticos , Enfermedades de las Plantas/virología , Virus ARN/crecimiento & desarrollo , Virus ARN/genética , Brasil , Genes Reporteros , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Oxidorreductasas/análisis , Oxidorreductasas/genética , Virus ARN/aislamiento & purificación , Genética Inversa , Coloración y Etiquetado , Tabaco/virología
7.
Chemistry ; 26(7): 1511-1517, 2020 Feb 03.
Artículo en Inglés | MEDLINE | ID: mdl-31867761

RESUMEN

Solid-state 19 F NMR is a powerful method to study the interactions of biologically active peptides with membranes. So far, in labelled peptides, the 19 F-reporter group has always been installed on the side chain of an amino acid. Given the fact that monofluoroalkenes are non-hydrolyzable peptide bond mimics, we have synthesized a monofluoroalkene-based dipeptide isostere, Val-Ψ[(Z)-CF=CH]-Gly, and inserted it in the sequence of two well-studied antimicrobial peptides: PGLa and (KIGAKI)3 are representatives of an α-helix and a ß-sheet. The conformations and biological activities of these labeled peptides were studied to assess the suitability of monofluoroalkenes for 19 F NMR structure analysis.


Asunto(s)
Alquenos/química , Péptidos Catiónicos Antimicrobianos/química , Membrana Celular/química , Espectroscopía de Resonancia Magnética , Secuencia de Aminoácidos , Péptidos Catiónicos Antimicrobianos/síntesis química , Flúor/química , Conformación Proteica en Hélice alfa , Coloración y Etiquetado/métodos
8.
Eur J Histochem ; 63(4)2019 Dec 12.
Artículo en Inglés | MEDLINE | ID: mdl-31833331

RESUMEN

The main step in the assessment of nanomaterial safety and suitability for biomedical use is the location and the dynamic tracking of nanoparticles (NPs) inside cells or tissues. To precisely investigate the uptake mechanisms and intracellular fate of NPs, transmission electron microscopy is the technique of choice; however, the detection of NPs may sometimes be problematic. In fact, while NPs containing strongly electron dense (e.g., metal) components do not require specific detection methods at the ultrastructural level, organic NPs are hardly detectable in the intracellular environment due to their intrinsic moderate electron density. In this study, the critical-electrolyte-concentration Alcian Blue method set up by Schofield et al. in 1975 was applied to track hyaluronic-acid-based NPs in muscle cells in vitro. This long-established histochemical method proved to be a powerful tool allowing to identify not only whole NPs while entering cells and moving into the cytoplasm, but also their remnants following lysosomal degradation and extrusion.


Asunto(s)
Azul Alcián/química , Colorantes/química , Ácido Hialurónico/metabolismo , Nanopartículas/metabolismo , Animales , Línea Celular , Lisosomas/metabolismo , Ratones , Microscopía Electrónica de Transmisión/métodos , Mioblastos/ultraestructura , Coloración y Etiquetado
9.
Indian J Dent Res ; 30(5): 703-707, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31854360

RESUMEN

Background: Oral cytology studies have claimed that cytoplasmic Periodic Acid Schiff (PAS) positivity in type-2 diabetics is due to glycogen content. But, it can also be due to mucin and glycoconjugates. Aim: 1. To confirm that cytoplasmic PAS positivity in type-2 diabetics is due to glycogen using diastase. 2. To know the effect of diabetes by determining the number of glycogen-containing cells in the smear. 3. To assess the impact of duration of diabetes based on PAS staining of cells. 4. To correlate between random blood glucose level and the number of PAS-positive cells. Materials and Methods: Study population comprised 45 individuals with 30 type-2 diabetics as case group (Group I < 5 years duration; Group II > 5 years duration) and 15 healthy volunteers (age and gender-matched) as control. For all subjects, random blood glucose was estimated and two cytosmears were obtained. The smears were stained with PAS and PAS-diastase stains (PAS-D). Staining intensity was documented as score 1 (mild-to-moderate) and score 2 (moderate-to-intense) and data obtained were statistically analyzed in SPSS version 16.0. Results: Mann-Whitney U test revealed that in diabetics cytoplasmic PAS positivity is because of glycogen (P < 0.05). There is an increase in the number of glycogen-containing cells (P < 0.05) in diabetics. The duration of diabetes had less impact on intracellular glycogen accumulation (P > 0.05). Spearman's correlation test revealed no significant correlation (P > 0.05) between random blood glucose and a number of PAS-positive cells. Conclusion: PAS positivity is because of intracellular glycogen accumulation in type-2 diabetics. It can convey the glycaemic status of an individual in the recent past, thus a beneficial role in screening and therapeutic monitoring.


Asunto(s)
Diabetes Mellitus , Glucógeno , Glucemia , Colorantes , Humanos , Reacción del Ácido Peryódico de Schiff , Coloración y Etiquetado
10.
Bratisl Lek Listy ; 120(11): 839-842, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31747764

RESUMEN

INTRODUCTION: Although the immunohistochemical staining with cytokeratin 7 (CK7) is an adjuvant method for identifying various components of the intrahepatic biliary system, the expression of CK7 does not occur in hepatocytes. In the literature, some studies suggest that a group of cells having dual morphologic and immunophenotypic characteristics of bile duct epithelium and hepatocytes, referred to as progenitor stem cells, was stained positive with CK7. MATERIALS AND METHODS: In this study, we examined a total of 219 cases diagnosed with autoimmune hepatitis, chronic hepatitis B, chronic hepatitis C, and primary biliary cholangitis between 2005 and 2017 in Uludag University, Faculty of Medicine, Department of Medical Pathology. RESULTS: The comparisons of AIH cases with HepB, HepC and PBC cases demonstrated that the immunoreactivity to CK7 was significantly higher in the AIH group (p < 0.005) compared to the groups of HepB and HepC, whereas no significant differences were found between the AIH and PBC groups. CONCLUSIONS: In our study, it was concluded that the immunoreactivity to CK7 could be used as an adjuvant treatment to the clinicopathologic assessment in distinguishing between the AIH cases and chronic viral hepatitis. However, since CK7 immunoreactive hepatocytes were widely detected also in patients with chronic viral hepatitis, and there was no statistically significant difference between the PBC and AIH cases, it has been established that the inclusion of CK7 immunoreactivity into the diagnostic histopathological criteria for AIH would not be convenient (Tab. 1, Fig. 1, Ref. 22).


Asunto(s)
Hepatitis Autoinmune/diagnóstico , Inmunohistoquímica , Queratina-7/análisis , Diagnóstico Diferencial , Hepatitis B Crónica , Hepatitis C Crónica , Humanos , Cirrosis Hepática Biliar , Coloración y Etiquetado
11.
Am J Forensic Med Pathol ; 40(4): 304-311, 2019 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-31687979

RESUMEN

Semen is crucial evidence for some sex crimes, with its sole confirmation being sperm detection. The success of sperm detection is dependent on all levels of preanalytic and analytic procedures. Specimen collection must be performed by well-trained and competent forensic physicians as well as forensic nurses, with preservation done properly before laboratory transfer. Laboratory procedures should consider archival sperm identification, by visualization, with adequate amounts separated from other cells to obtain male DNA profiles. Differential extraction is robust and accepted as the forensic standard but is time consuming and may result in male DNA loss. Thus, alternative methods and microdevices have been developed. Challenges in sperm isolation from vaginal or buccal epithelium mixes and discrimination in multiperpetrator cases have been overcome by single-cell profiling; however, problems inherent in identical twin discrimination and azoospermia have yet to be solved. Epigenetics and future molecular biomarkers may hold the key; therefore, all laboratory processes must consider DNA and RNA protection. Long-term specimen preservation should be done when possible in light of future confirmatory tests.


Asunto(s)
Ciencias Forenses/métodos , Manejo de Especímenes , Espermatozoides/citología , Separación Celular , Dermatoglifia del ADN , Metilación de ADN , Femenino , Humanos , Captura por Microdisección con Láser , Masculino , Repeticiones de Microsatélite , Reacción en Cadena de la Polimerasa/métodos , Antígeno Prostático Específico/aislamiento & purificación , ARN Mensajero/aislamiento & purificación , Proteínas de Secreción de la Vesícula Seminal/aislamiento & purificación , Delitos Sexuales , Coloración y Etiquetado , Factores de Tiempo
12.
Klin Lab Diagn ; 64(10): 632-634, 2019.
Artículo en Ruso | MEDLINE | ID: mdl-31742958

RESUMEN

Current methods of biofilm imaging do not support a differentiated assessment of its composition, since it is not possible to establish a substrate stained with crystal violet, as this dye can form complexes with both intracellular and extracellular structures. This approach does not adequately assess the anti-biofilm effects of drugs, while the results of studying the interaction of drugs with biofilm components can ensure their most correct choice. The aim of investigation was to study the possibility of applying the original modification of the current method to determine the ratio of the cellular part and the matrix of biofilms of gram-positive microorganisms. The biofilm components were analyzed using a two-step approach, when prepared biofilms of gram-positive microorganisms were stained with crystal violet for 5 minutes, followed by fixing the dye in bacterial cells with iodine solution, and then the colored products were dissolved with 95% alcohol: matrix components for 1 minute, total biofilm for 15 minutes, after which the composition of biofilms was estimated by the formula: M=(OP1/OP15)×100, Kb=100-M, where M is the proportion of the matrix,%; Kb - the proportion of the cellular component,%; OP1 - optical density of samples, when alcohol was allowed to dissolve the colored product for no more than 1 minute; OP15 - was the optical density of samples, when alcohol is allowed to dissolve the colored product for 15 minutes. It was shown that in the composition of the biofilm formed by the collection strain, the proportion of the matrix was 13.2%, and the cellular component accounted for 86.8%. When the same strain cultivated in the presence of an antibiotic, an increase in the biofilm matrix was observed, which is probably due to the compensatory response of the microorganism to the action of the antibiotic. The proposed approach to the study of biofilms makes it possible to evaluate its component composition. Obtaining additional information in this way can provide, inter alia, an increase in the effectiveness of antimicrobial therapy while reducing the study time.


Asunto(s)
Biopelículas , Bacterias Grampositivas/clasificación , Coloración y Etiquetado
13.
Indian J Dent Res ; 30(4): 568-572, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31745054

RESUMEN

Context: The aims of this study were (i) to evaluate the color stability of two orthodontic adhesives and (ii) to evaluate the color stability of enamel and ceramic brackets bonded with orthodontic adhesives after exposure to different staining agents. Materials and Methods: Disks were prepared with two orthodontic adhesives (Transbond and Enlight). Color stability evaluation was performed with a spectrophotometer using CIELab parameters. The specimens were divided into four groups and immersed in the following staining agents (n = 5): distilled water (control), coffee, red wine, and cola soft drink, for 1 h/day for 120 days. Twenty molar crowns were also used. The baseline color of enamel was obtained and ceramic brackets were bonded with the orthodontic adhesives. The enamel specimens were divided into four groups and immersed in the same staining agents. After 120 days, another color reading with the brackets in position was taken. The brackets were then removed and the enamel color was again evaluated. Color difference (ΔE) in different time periods was determined and the data were analyzed by ANOVA and Tukey's test (α = 5%). Results: Transbond showed lower ΔE than Enlight. Water, cola, and coffee had the lowest ΔE values. Immersion in wine showed the highest ΔE values. For time, the lower ΔE values were found for 24 h and 7 days. Storage times of 60, 90, and 120 days showed the highest ΔE values. ΔE for enamel showed significant differences only for time. Conclusion: Adhesive, staining agents, and storage time influenced the color stability of orthodontic adhesives.


Asunto(s)
Cementos Dentales , Soportes Ortodóncicos , Color , Esmalte Dental , Ensayo de Materiales , Cementos de Resina , Coloración y Etiquetado , Propiedades de Superficie
14.
Water Sci Technol ; 80(5): 817-826, 2019 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-31746788

RESUMEN

Accurate evaluation of viable Ascaris ova in wastewater is the key to mitigating Ascaris reinfections in endemic regions. In this study, the viability of Ascaris ova in raw wastewater was determined using three different detection methods: culture-based, BacLight Live/Dead staining and propidium monoazide-quantitative polymerase chain reaction (PMA-qPCR). Furthermore, comparative assessment of viability utilising the aforementioned detection methods was performed using seeded experiments in wastewater. The percentage of viability was: culture-based (82%), BacLight Live/Dead staining (87%) and PMA-qPCR (85%) respectively. Despite the fact that no statistical difference was shown in the viability determination among the three methods, PMA-qPCR-based viability determination would be preferable over the other two methods for evaluating potential public health risks with A. suum ova due to its accuracy, being least subjective and its rapid reaction time.


Asunto(s)
Ascaris , Aguas Residuales , Animales , Azidas , Viabilidad Microbiana , Propidio , Reacción en Cadena en Tiempo Real de la Polimerasa , Coloración y Etiquetado
15.
Nan Fang Yi Ke Da Xue Xue Bao ; 39(9): 1038-1044, 2019 Sep 30.
Artículo en Chino | MEDLINE | ID: mdl-31640956

RESUMEN

OBJECTIVE: To optimize the method for embedding multiple undecalcified mouse tibias in plastic blocks, improve the efficiency and stability of plastic embedding and reduce the detachment rate of plastic slides. METHODS: Thirty undecalcified tibias from 15 B6 mice were used for plastic embedding after calcein labeling, fixation, dehydration and infiltration. The tibias were embedded in cylindrical plastic blocks with a diameter of 4 mm. For each bone, the 1/4 proximal tibia was cut off, and the remaining 3/4 was used for re-embedding. Five bones were embedded in a single block with each bone standing closely on the surface of a flat plate. The samples were randomized into control and experimental groups in all the processes of embedding, sectioning and staining. In the 3 groups with modified embedment, flowing CO2 was added into the embedding solution, embedding solution was applied to the section surface, and the slides were heated at 95 ℃ for 15 min. The polymerization time, slide detachment rate, bone formation and osteoblast parameters were analyzed. RESULTS: We prepared 6 plastic blocks, each containing 5 tibias, whose cross sections were on the same plane. The blocks were completely polymerized and suitable for sectioning. Flowing CO2 into the embedding solution reduced the polymerization time and increased the rate of complete polymerization. Application of the embedding solution on the section surface significantly reduced the detachment rate of the sections (P < 0.05) without affecting bone formation analysis (P > 0.05). Heating the slides significantly lowered the detachment rate of the sections (P < 0.05) without affecting osteoblast analysis (P > 0.05). CONCLUSIONS: The optimized method allows effective embedding of multiple undecalcified mice tibias in the same block and can be an ideal method for histological analysis of undecalcified bones.


Asunto(s)
Plásticos , Tibia , Adhesión del Tejido/métodos , Animales , Ratones , Coloración y Etiquetado
16.
Chem Commun (Camb) ; 55(85): 12865-12868, 2019 Nov 04.
Artículo en Inglés | MEDLINE | ID: mdl-31599283

RESUMEN

4-Oxime-1,8-naphthalimide was reported as a novel bioorthogonal turn-on probe based on 1,3-dipolar cycloaddition reactions between in situ generated nitrile oxide and alkenes/alkynes. The resulting isoxazole products displayed dramatically strong fluorescence enhancement upon photoirradiation through isoxazole-oxazole photoisomerization. This new methodology was successfully applied for in situ fluorogenic protein labeling.


Asunto(s)
Colorantes Fluorescentes/química , Naftalimidas/química , Oximas/química , Proteínas/química , Reacción de Cicloadición , Coloración y Etiquetado
17.
Nat Commun ; 10(1): 4697, 2019 10 16.
Artículo en Inglés | MEDLINE | ID: mdl-31619683

RESUMEN

Comprehensive protein identification and concomitant structural probing of proteins are of great biological significance. However, this is challenging to accomplish simultaneously in one confined space. Here, we develop a nanosecond photochemical reaction (nsPCR)-based click chemistry, capable of structural probing of proteins and enhancing their identifications through on-demand removal of surrounding matrices within nanoseconds. The nsPCR is initiated using a photoactive compound, 2-nitrobenzaldehyde (NBA), and is examined by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). Benefiting from the on-demand matrix-removal effect, this nsPCR strategy enables enhanced neuropeptide identification and visualization from complex tissue samples such as mouse brain tissue. The design shows great promise for structural probing of proteins up to 155 kDa due to the exclusive accessibility of nsPCR to primary amine groups, as demonstrated by its general applicability using a series of proteins with various lysine residues from multiple sample sources, with accumulated labeling efficiencies greater than 90%.


Asunto(s)
Encéfalo/metabolismo , Química Clic , Neuropéptidos/metabolismo , Procesos Fotoquímicos , Coloración y Etiquetado , Animales , Benzaldehídos , Braquiuros , Ratones , Imagen Óptica , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción
18.
J Helminthol ; 94: e95, 2019 Sep 30.
Artículo en Inglés | MEDLINE | ID: mdl-31564254

RESUMEN

There is geographical variation in the morphology and genetics of Wuchereria bancrofti, the major cause of human lymphatic filariasis. This study aims to compare morphological and genetic variation of W. bancrofti microfilariae recovered from carriers in Lao PDR, Myanmar and Thailand. Six morphological parameters (body length, cephalic space length and width, length of head to nerve ring, body width at nerve ring, Innenkȍrper length and number of column nuclei between the cephalic space and nerve ring) were evaluated from microfilariae in Giemsa-stained thick blood films. A portion of the cytochrome c oxidase subunit 1 gene of mitochondrial DNA was sequenced and analysed. Wuchereria bancrofti microfilariae showed a wide variation in their morphology and morphometry among three countries. Phylogenetic analysis confirmed that all microfilariae belonged to W. bancrofti. Higher mutation frequencies were observed in samples from Myanmar, relative to Thailand and Lao PDR. This study highlights the morphological disparities of microfilariae and genetic variability within W. bancrofti among three geographical locations. We found that reported morphometric differences between localities were less clear-cut than previously thought. Further studies are needed to determine the microfilarial periodicity in Lao PDR.


Asunto(s)
Portador Sano/parasitología , Filariasis Linfática/parasitología , Variación Genética , Wuchereria bancrofti/crecimiento & desarrollo , Wuchereria bancrofti/aislamiento & purificación , Animales , Colorantes Azulados/química , Sangre/parasitología , Femenino , Laos , Masculino , Microfilarias/clasificación , Microfilarias/genética , Microfilarias/crecimiento & desarrollo , Microfilarias/aislamiento & purificación , Mutación , Filogenia , Coloración y Etiquetado , Tailandia , Wuchereria bancrofti/clasificación , Wuchereria bancrofti/genética
19.
Nihon Yakurigaku Zasshi ; 154(4): 165-170, 2019.
Artículo en Japonés | MEDLINE | ID: mdl-31597894

RESUMEN

Immunofluorescence microscopy is a powerful method for analysis of the subcellular localization of the protein of interest. The use of fluorescence is very effective for multiple labeling and for higher magnification observation with a laser confocal microscope. A basic protocol of the immunofluorescent staining in cultured cells with some useful suggestions are introduced in the present paper. The author describes the following contents. 1) Coverslips: coverslips are necessary to be coated with coating reagent such as collagen to improve the attachment and growing of the cells. Commercially available glass slides for cell culture are also useful. Permeable support filters are convenient for establishing the apical and basolateral compartments when epithelial cells are cultured. 2) Fixation: basic fixative is 4% paraformaldehyde in phosphate buffer. Ethanol including 1% acetic acid or pure ethanol or methanol are also effective for some antigens. 3) Permeabilization: treatment with Triton X-100 or saponin before and sometimes during the antibody incubation is required to improve the antibody accessibility. 4) Blocking: nonspecific binding of antibodies is blocked with 5% serum from the animal species as same as that of the secondary antibody you use. 5) Antibody incubations: antibodies are diluted in the blocking solution and incubated with samples. For multi-labeling with primary antibodies derived from different animal species, the specimen can be sequentially incubated with a mixture of primary antibodies and a mixture of secondary antibodies. 6) Choice of secondary antibodies: secondary antibodies which do react specifically to the target without cross-reaction and appropriate fluorescent dyes for multiple labeling are required. 7) Sample storage: the fluorescence can be kept for long period like several months to years in specimens which are mounted with anti-fading mounting medium and stored at -20°C. 8) Trouble shootings: some trouble shootings are also shown. The author hopes that this paper would help the readers to obtain better results in immunofluorescence.


Asunto(s)
Técnica del Anticuerpo Fluorescente , Microscopía Fluorescente , Animales , Anticuerpos , Células Cultivadas , Coloración y Etiquetado
20.
Chemistry ; 25(61): 13994-14002, 2019 Nov 04.
Artículo en Inglés | MEDLINE | ID: mdl-31506999

RESUMEN

Quinone methide (QM) as a latent trapping unit has been widely explored in activity-based self-immobilizing reagents. However, further application of this strategy has been largely hampered by the limited labeling efficiency to proteins. In this study, a thorough investigation on the labeling efficiency and the structure of QM-based trapping unit is presented, from which a QM with multiple leaving groups was identified as an optimal trapping unit. An alkaline phosphatase (ALP) immobilizing reagent featured with this multiple-labeling trapping unit exhibited lower nonspecific binding and, remarkably, a significantly higher labeling efficiency over other immobilizing reagents upon enzymatic activation. The utility of this imaging reagent was further demonstrated with the in vitro and in vivo visualization of the ALP activities. Furthermore, the multiple functional trapping unit may find greater value in the other activity-based immobilizing probes.


Asunto(s)
Fosfatasa Alcalina/metabolismo , Indolquinonas/química , Fosfatasa Alcalina/química , Animales , Colorantes Fluorescentes/química , Células HEK293 , Células HeLa , Humanos , Indolquinonas/metabolismo , Ratones , Ratones Desnudos , Microscopía Fluorescente , Imagen Óptica , Coloración y Etiquetado , Trasplante Heterólogo
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