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1.
Curr Protoc ; 1(9): e239, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34495576

RESUMO

Advanced immunohistochemical (IHC) protocols aim to visualize different molecules in situ simultaneously. These techniques are of utmost importance as a first step in studying possible interactions of proteins at the subcellular level. Colocalized stains in tissue sections indicate proximity of two proteins of interest. Frequently, double staining protocols are restricted by the lack of primary antibodies generated in different animal species for indirect IHC visualization. Here, we present a detailed protocol for mouse inner ear tissue using two different primary rabbit antibodies directed against transmembrane ion channel proteins of cochlear neurons. The two antibodies are combined for fluorescence (confocal) as well as dual multiplex colorimetric visualization in two sequential single IHC stainings. A heat-denaturation step is performed in between. Primary antibody specificity is tested by preadsorption with the immunogenic peptide, and positive and negative tissue controls are performed to confirm the reliability of the antibody detection. We describe the whole procedure in detail beginning with tissue extraction of the mouse inner ear and continuing with chemical fixation, cryoembedding, and preparation for manual and fully automated immunostaining, including steps for heat-induced antigen retrieval. The potential to use antibodies from the same host species for single and double IHC staining opens up multiple possibilities for detecting different targets in the same tissue section using resources and materials that are widely available. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Tissue preparation, cryoembedding, and sectioning Basic Protocol 2: Double colorimetric immunostaining with an automatic immunostainer Basic Protocol 3: Double manual fluorometric immunostaining with fluorescence.


Assuntos
Cóclea , Temperatura Alta , Animais , Imuno-Histoquímica , Camundongos , Coelhos , Reprodutibilidade dos Testes , Coloração e Rotulagem
2.
Nat Commun ; 12(1): 4855, 2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34381044

RESUMO

The vertebrate brain consists of diverse neuronal types, classified by distinct anatomy and function, along with divergent transcriptomes and proteomes. Defining the cell-type specific neuroproteomes is important for understanding the development and functional organization of neural circuits. This task remains challenging in complex tissue, due to suboptimal protein isolation techniques that often result in loss of cell-type specific information and incomplete capture of subcellular compartments. Here, we develop a genetically targeted proximity labeling approach to identify cell-type specific subcellular proteomes in the mouse brain, confirmed by imaging, electron microscopy, and mass spectrometry. We virally express subcellular-localized APEX2 to map the proteome of direct and indirect pathway spiny projection neurons in the striatum. The workflow provides sufficient depth to uncover changes in the proteome of striatal neurons following chemogenetic activation of Gαq-coupled signaling cascades. This method enables flexible, cell-type specific quantitative profiling of subcellular proteome snapshots in the mouse brain.


Assuntos
Ascorbato Peroxidases/metabolismo , Núcleo Celular/metabolismo , Corpo Estriado/metabolismo , Proteoma/metabolismo , Animais , Ascorbato Peroxidases/genética , Corpo Estriado/citologia , Citosol/metabolismo , Espectrometria de Massas , Camundongos , Vias Neurais , Neurônios/citologia , Neurônios/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Coloração e Rotulagem , Fluxo de Trabalho
3.
Am J Physiol Cell Physiol ; 321(3): C607-C614, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34378992

RESUMO

Bovine milk exosomes (BMEs) are being explored in drug delivery despite their rapid elimination by macrophages. We aimed at identifying the BME transporter in murine bone marrow-derived macrophages (BMDMs). Fluorophore-labeled BMEs were used in transport studies in BMDMs from C57BL/6J and class A scavenger receptor type 1/2 (CASR-1/2) knockout mice and tissue accumulation in macrophage-depleted C57BL/6J mice. Parametric and nonparametric statistics tests for pairwise and multiple comparisons were used. Chemical inhibitors of phagocytosis by cytochalasin D led to a 69 ± 18% decrease in BME uptake compared with controls (P < 0.05), whereas inhibitors of endocytic pathways other than phagocytosis had a modest effect on uptake (P > 0.05). Inhibitors of class A scavenger receptors (CASRs) including CASR-1/2 caused a 70% decrease in BME uptake (P < 0.05). The uptake of BMEs by BMDMs from CASR-1/2 knockout mice was smaller by 58 ± 23% compared with wild-type controls (P < 0.05). Macrophage depletion by clodronate caused a more than 44% decrease in BME uptake in the spleen and lungs (P < 0.05), whereas the decrease observed in liver was not statistically significant. In conclusion, CASR-1/2 facilitates the uptake of BMEs in BMDMs and C57BL/6J mice.


Assuntos
Exossomos/metabolismo , Macrófagos/metabolismo , Leite/química , Receptores Depuradores Classe A/genética , Animais , Bovinos , Ácido Clodrônico/farmacologia , Citocalasina D/farmacologia , Endocitose/efeitos dos fármacos , Exossomos/química , Feminino , Corantes Fluorescentes/química , Expressão Gênica , Fígado/efeitos dos fármacos , Fígado/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fagocitose/efeitos dos fármacos , Isoformas de Proteínas/deficiência , Isoformas de Proteínas/genética , Receptores Depuradores Classe A/deficiência , Baço/efeitos dos fármacos , Baço/metabolismo , Coloração e Rotulagem/métodos
4.
Talanta ; 234: 122689, 2021 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-34364487

RESUMO

Beta-galactosidase (ß-gal) activity is closed related with senescence cells and aging-associated diseases, however, the traditional readout of ß-gal activity based on X-gal staining was limited to low sensitivity in short incubation times and false positives in long incubation times. Here, we expose the potential role of insoluble X-gal hydrolysates in causing false positives by diffusion pollution depending on organic medium and then propose the in-situ Surface-enhanced Raman spectroscopy (SERS) readout strategy to identify and locate ß-gal positive cells. By building the blue-white screening model and fabricating SERS-active needle sensor, the sensitive detection of ß-gal has been realized with the detection limit of less than 1 nmol L-1. The in-situ SERS readout strategy is proved to be necessary and feasible to improve the reliability of X-gal staining assay through shortening the time to a few hours. Moreover, its application was also preliminarily evaluated to analyse individual cells and tissues, which showed the well consistency for judgement of ß-gal activity cells at different times. Consequently, by improving reliability and reducing time consumption, this SERS readout strategy may be of great significance to promote the application of X-gal staining assay in biology and biomedicine.


Assuntos
Galactosídeos , Indóis , Reprodutibilidade dos Testes , Coloração e Rotulagem , beta-Galactosidase
5.
Am J Dent ; 34(4): 205-210, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34370913

RESUMO

PURPOSE: To evaluate the rehardening ability of SDF and its individual components, silver, and fluoride ions, on early enamel caries lesions using artificial saliva with and without mucin. METHODS: Early caries lesions were created in human permanent enamel specimens. The specimens (n=36 per group) were then treated with a single application of: SDF (38%), SDF followed by application of potassium iodide (SDF+KI), potassium fluoride (KF); fluoride control, 44,800 ppm (F), silver nitrate (AgNO3); silver control, 253,900 ppm (Ag), or deionized water (DIW). Immediately, the specimens were subjected to 4 days of continuous remineralization with or without mucin (n=18 per subgroup). Changes in Vickers surface microhardness from lesion baseline (ΔVHN) were calculated. Data were analyzed using two-way (intervention vs. rehardening models) ANOVA. RESULTS: In both rehardening models (with or without mucin), SDF (ΔVHN data; mean ± standard deviation; with/without mucin: 26± 19/3± 11) was significantly less effective in rehardening promotion than SDF+KI (37± 12/39± 16) and KF (40± 17/41± 29; P≤ 0.0332). Compared to AgNO3 (9± 9/18± 15) and DIW (3± 7/12± 9), SDF was more effective in the presence of mucin (P≤ 0.001) but not in its absence, similar to DIW (P= 0.1117); less effective vs. AgNO3 (P= 0.0061). The presence of mucin significantly increased the rehardening ability of SDF (P< 0.0001). However, mucin did not affect the extent of rehardening in the other groups (P≥ 0.082). SDF+KI and KF were superior in their ability in rehardening promotion than AgNO3 and DIW in both rehardening models (P< 0.0001). In both rehardening models, ΔL* values from baseline to post-rehardening show that applying KI after SDF significantly lessened the dark staining caused by SDF (P< 0.0001). Under the present in vitro conditions, SDF does not appear to enhance surface rehardening of early enamel caries lesions. The co-presence of mucin during rehardening enhanced the efficacy of SDF which warrants further investigation. CLINICAL SIGNIFICANCE: Silver diamine fluoride + potassium iodide may be a viable option in rehardening of early enamel caries lesions.


Assuntos
Cárie Dentária , Mucinas , Cariostáticos , Cárie Dentária/tratamento farmacológico , Cárie Dentária/prevenção & controle , Suscetibilidade à Cárie Dentária , Fluoretos Tópicos , Humanos , Compostos de Amônio Quaternário , Compostos de Prata , Fluoreto de Sódio , Coloração e Rotulagem
6.
Vestn Oftalmol ; 137(4): 18-23, 2021.
Artigo em Russo | MEDLINE | ID: mdl-34410052

RESUMO

PURPOSE: To develop a method of viscodissection involving staining of epiretinal membranes, and to evaluate its efficacy and safety in surgical treatment of proliferative diabetic retinopathy (PDR). MATERIAL AND METHODS: The study included 30 patients with type 1 diabetes mellitus and PDR with tractional retinal detachment (TRD). All patients were divided into two groups. In the first (main) group, at the initial stages of the operation, viscodissection with staining of epiretinal structures was performed, followed by segmentation and removal of membranes; in the second (control) group, segmentation and removal of membranes was performed using a vitreotome and endovitreal forceps. RESULTS: During the follow-up, all patients showed positive trends of morphological and functional indicators. While the number of intraoperative stages was the same in both groups, the total operation time in patients of the main group was significantly lower (p≤0.001) than in patients of the control group (main - 41.3±2.8 min; control - 53.8±6.2 min). With equal number of posterior hyaloid membrane to inner limiting membrane (PHM to ILM) fixation points in both groups, iatrogenic retinal rupture occurred significantly less frequently in patients of the main group (main - 0.6±0.7, control - 3.1±2.9) (p≤0.001). In this regard, among the patients of the control group, in the overwhelming majority of cases, it was necessary to use a silicone oil tamponade (60%) or gas-air mixture (33%), while in the first group the main postoperative media were sterile BSS solution (73%) and gas-air mixture (27%).


Assuntos
Diabetes Mellitus , Retinopatia Diabética , Membrana Epirretiniana , Descolamento Retiniano , Retinopatia Diabética/diagnóstico , Retinopatia Diabética/cirurgia , Membrana Epirretiniana/diagnóstico , Membrana Epirretiniana/cirurgia , Humanos , Descolamento Retiniano/diagnóstico , Descolamento Retiniano/etiologia , Descolamento Retiniano/cirurgia , Coloração e Rotulagem , Vitrectomia
7.
Molecules ; 26(16)2021 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-34443576

RESUMO

Bioorthogonal click-reactions represent ideal means for labeling biomolecules selectively and specifically with suitable small synthetic dyes. Genetic code expansion (GCE) technology enables efficient site-selective installation of bioorthogonal handles onto proteins of interest (POIs). Incorporation of bioorthogonalized non-canonical amino acids is a minimally perturbing means of enabling the study of proteins in their native environment. The growing demand for the multiple modification of POIs has triggered the quest for developing orthogonal bioorthogonal reactions that allow simultaneous modification of biomolecules. The recently reported bioorthogonal [4 + 1] cycloaddition reaction of bulky tetrazines and sterically demanding isonitriles has prompted us to develop a non-canonical amino acid (ncAA) bearing a suitable isonitrile function. Herein we disclose the synthesis and genetic incorporation of this ncAA together with studies aiming at assessing the mutual orthogonality between its reaction with bulky tetrazines and the inverse electron demand Diels-Alder (IEDDA) reaction of bicyclononyne (BCN) and tetrazine. Results showed that the new ncAA, bulky-isonitrile-carbamate-lysine (BICK) is efficiently and specifically incorporated into proteins by genetic code expansion, and despite the slow [4 + 1] cycloaddition, enables the labeling of outer membrane receptors such as insulin receptor (IR) with a membrane-impermeable dye. Furthermore, double labeling of protein structures in live and fixed mammalian cells was achieved using the mutually orthogonal bioorthogonal IEDDA and [4 + 1] cycloaddition reaction pair, by introducing BICK through GCE and BCN through a HaloTag technique.


Assuntos
Código Genético , Lisina/química , Lisina/genética , Nitrilas/química , Reação de Cicloadição , Corantes Fluorescentes/química , Coloração e Rotulagem
8.
Int J Mol Sci ; 22(16)2021 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-34445289

RESUMO

The NSs protein and the nucleocapsid protein (NP) of orthotospoviruses are the major targets for serological detection and diagnosis. A common epitope of KFTMHNQIF in the NSs proteins of Asia orthotospoviruses has been applied as an epitope tag (nss-tag) for monitoring recombinant proteins. In this study, a monoclonal antibody TNP MAb against the tomato spotted wilt virus (TSWV) NP that reacts with TSWV-serogroup members of Euro-America orthotospoviruses was produced. By truncation and deletion analyses of TSWV NP, the common epitope of KGKEYA was identified and designated as the np sequence. The np sequence was successfully utilized as an epitope tag (np-tag) to monitor various proteins, including the green fluorescence protein, the coat protein of the zucchini yellow mosaic virus, and the dust mite chimeric allergen Dp25, in a bacterial expression system. The np-tag was also applied to investigate the protein-protein interaction in immunoprecipitation. In addition, when the np-tag and the nss-tag were simultaneously attached at different termini of the expressed recombinant proteins, they reacted with the corresponding MAbs with high sensitivity. Here, we demonstrated that the np sequence and TNP MAb can be effectively applied for tagging and detecting proteins and can be coupled with the nss-tag to form a novel epitope-tagging system for investigating protein-protein interactions.


Assuntos
Mapeamento de Epitopos , Imuno-Histoquímica/métodos , Proteínas do Nucleocapsídeo/imunologia , Vírus de Plantas/imunologia , América , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Epitopos/análise , Epitopos/química , Europa (Continente) , Imunoprecipitação , Vírus do Mosaico/química , Vírus do Mosaico/classificação , Vírus do Mosaico/imunologia , Proteínas do Nucleocapsídeo/química , Doenças das Plantas/imunologia , Doenças das Plantas/virologia , Vírus de Plantas/química , Vírus de Plantas/classificação , Potyvirus/química , Potyvirus/imunologia , Coloração e Rotulagem/métodos , Tospovirus/química , Tospovirus/classificação , Tospovirus/imunologia
9.
Int J Mol Sci ; 22(16)2021 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34445351

RESUMO

Multiplexed single-cell analysis of proteins in their native cellular contexts holds great promise to reveal the composition, interaction and function of the distinct cell types in complex biological systems. However, the existing multiplexed protein imaging technologies are limited by their detection sensitivity or technical demands. To address these issues, here, we develop an ultrasensitive and multiplexed in situ protein profiling approach by reiterative staining with off-the-shelf antibodies and cleavable fluorescent tyramide (CFT). In each cycle of this approach, the protein targets are recognized by antibodies labeled with horseradish peroxidase, which catalyze the covalent deposition of CFT on or close to the protein targets. After imaging, the fluorophores are chemically cleaved, and the antibodies are stripped. Through continuous cycles of staining, imaging, fluorophore cleavage and antibody stripping, a large number of proteins can be quantified in individual cells in situ. Applying this method, we analyzed 20 different proteins in each of ~67,000 cells in a human formalin-fixed paraffin-embedded (FFPE) tonsil tissue. Based on their unique protein expression profiles and microenvironment, these individual cells are partitioned into different cell clusters. We also explored the cell-cell interactions in the tissue by examining which specific cell clusters are selectively associating or avoiding each other.


Assuntos
Diagnóstico por Imagem/métodos , Proteínas/metabolismo , Análise de Célula Única/métodos , Anticorpos/metabolismo , Comunicação Celular , Imunofluorescência/métodos , Corantes Fluorescentes/química , Corantes Fluorescentes/farmacocinética , Formaldeído/química , Peroxidase do Rábano Silvestre/análise , Peroxidase do Rábano Silvestre/metabolismo , Humanos , Técnicas Imunoenzimáticas/métodos , Tonsila Palatina/química , Tonsila Palatina/citologia , Tonsila Palatina/metabolismo , Inclusão em Parafina , Proteínas/análise , Sensibilidade e Especificidade , Coloração e Rotulagem/métodos
10.
ACS Appl Mater Interfaces ; 13(33): 40070-40078, 2021 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-34387999

RESUMO

Aminothiols are closely related to chronic kidney disease, but little is known regarding levels of related aminothiols in the urine of immunoglobulin A vasculitis with nephritis (IgAVN) patients. Herein, a well-defined core-shell Zr-based metal-organic framework (Zr-MOF) composite SiO2@50Benz-Cys was constructed as a mercury ion affinity material via a solvent-assisted ligand exchange strategy for the selective extraction and enrichment of low-concentration aminothiols in IgAVN patient urine. SiO2@50Benz-Cys was competent to enrich the total glutathione (GSH) and total homocysteine (Hcy) in virtue of the excellent affinity after chelation with mercury ions. The extraction efficiencies were closely related to the pH, dithiothreitol amount, and the dose of functional Zr-MOF. Coupled with HPLC-MS/MS in optimized conditions, GSH and Hcy were determined with low detection limits of 0.5 and 1 nmol L-1, respectively. The recoveries of GSH and Hcy for the urine sample at three spiked levels were in the range of 85.3-105% and 79.5-103%, which showed good precision and accuracy. Benefiting from the matrix interference elimination in the process of extraction, the simultaneous detection of aminothiols in the urine of the healthy group and immunoglobulin A vasculitis (IgAV) and IgAVN patients was successfully carried out, suggesting that the Zr-MOF and the robust method together provided a potential application in the analysis of urinary biomolecules. The analysis of variance (ANOVA) showed that the levels of GSH and Hcy had significant differences between the patients and the control. This work is very valuable as it provides a better understanding of concentration alterations of GSH and Hcy in urine involved with IgAVN for clinical research.


Assuntos
Glutationa/urina , Homocisteína/urina , Estruturas Metalorgânicas/química , Nefrite/diagnóstico , Zircônio/química , Técnicas Biossensoriais , Cromatografia Líquida de Alta Pressão , Humanos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Dióxido de Silício , Coloração e Rotulagem/métodos , Compostos de Sulfidrila/química , Espectrometria de Massas em Tandem
11.
J Contemp Dent Pract ; 22(6): 691-702, 2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-34393129

RESUMO

AIM: Odontogenic tumors (OTs) and bone lesions of the oral cavity present diverse histological features and varying clinical behavior that makes predicting their biologic behavior difficult. The research undertaken in the current study aims to predict the biological behavior of oral hyalinizing odontogenic and bone lesions (OHO-BL) for the first time by employing four differential stains with clinicopathologic correlation. MATERIALS AND METHODS: The study was performed on retrospectively diagnosed formalin-fixed paraffin-embedded cases of OTs (n = 53) and bone lesions (n = 10). The severity of hyalinization (SOH) was assessed from stained tissue sections. Polarizing microscopy was used to analyze hyalinization in tissues stained with differential special stains, namely periodic acid-Schiff (PAS), Safranin-O, Alcian Blue, and Picrosirius red. SOH was also analyzed for possible correlation with recurrence and clinicopathologic correlation in OHO-BL. RESULTS: Intense staining was observed with PAS, Alcian Blue, and Safranin-O in OTs with increased SOH with a statistical significance. Polarizing greenish yellow color correlated significantly with the recurrence potential of the OT group. Recurrence in individual lesions of the OT group showed a statistically significant association with SOH. Such individual correlation was not observed in bone diseases. CONCLUSION: PAS, Alcian Blue, Safranin-O, and Picrosirius red are reliable stains to assess hyalinization in OHO-BL. Picrosirius red-polarizing microscopy is a dependable tool for identifying recurrent odontogenic lesions. CLINICAL SIGNIFICANCE: SOH can be considered a histological predictor of aggressive biologic behavior in oral hyalinizing odontogenic lesions that can enable the surgeon to arrive at an appropriate management protocol.


Assuntos
Doenças Ósseas , Tumores Odontogênicos , Corantes , Humanos , Microscopia de Polarização , Recidiva Local de Neoplasia , Estudos Retrospectivos , Coloração e Rotulagem
12.
Nat Commun ; 12(1): 4884, 2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34385460

RESUMO

Pathology is practiced by visual inspection of histochemically stained tissue slides. While the hematoxylin and eosin (H&E) stain is most commonly used, special stains can provide additional contrast to different tissue components. Here, we demonstrate the utility of supervised learning-based computational stain transformation from H&E to special stains (Masson's Trichrome, periodic acid-Schiff and Jones silver stain) using kidney needle core biopsy tissue sections. Based on the evaluation by three renal pathologists, followed by adjudication by a fourth pathologist, we show that the generation of virtual special stains from existing H&E images improves the diagnosis of several non-neoplastic kidney diseases, sampled from 58 unique subjects (P = 0.0095). A second study found that the quality of the computationally generated special stains was statistically equivalent to those which were histochemically stained. This stain-to-stain transformation framework can improve preliminary diagnoses when additional special stains are needed, also providing significant savings in time and cost.


Assuntos
Biópsia com Agulha de Grande Calibre/métodos , Aprendizado Profundo , Diagnóstico por Computador/métodos , Nefropatias/patologia , Rim/patologia , Coloração e Rotulagem/métodos , Algoritmos , Corantes/química , Corantes/classificação , Corantes/normas , Diagnóstico Diferencial , Humanos , Nefropatias/diagnóstico , Patologia Clínica/métodos , Patologia Clínica/normas , Padrões de Referência , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Coloração e Rotulagem/normas
13.
Arkh Patol ; 83(4): 36-44, 2021.
Artigo em Russo | MEDLINE | ID: mdl-34278759

RESUMO

Kikuchi-Fujimoto disease (KFD) is a rare disease that is clinically manifested mainly by fever and lymphadenopathy. KFD was originally believed to occur primarily in East Asia women, this disease was subsequently described in all ethnic groups worldwide. The important differential diagnostic feature of KFD is the detection of CD123-expressing plasmocytoid dendritic cells (PDCs) in the tissue of the affected lymph node. The standard immunohistochemical staining method has sufficient sensitivity and specificity to detect CD123, but it gives no way of judging the possible phenotypic heterogeneity of cells with CD123 expression. OBJECTIVE: To identify the phenotypic heterogeneity of CD123-expressing cells in the affected lymph nodes in patients with KFD by a sequential immunoperoxidase labeling and erasing (SIMPLE) method. MATERIAL AND METHODS: Excision biopsies of lymph nodes were examined in 3 patients with KFD. After an immunohistochemical reaction using a single antibody, the tissue specimen was digitized with a Pannoramic 250 Flash III scanner (3DHISTECH, Hungary), then the cover glass was removed from the section, the specimen was hydrated and placed in a specialized buffer. Then the following primary antibody was applied to the washed tissue specimen and further immunohistochemical reaction and scanning were performed. As a result, each tissue specimen was sequentially stained in reactions with 4 antibodies. The microphotographs of specimens stained in a reaction with anti-CD123 antibody showed positive cells for their identification in the Pannoramic Viewer program (3DHISTECH, Hungary) on the remaining microphotographs displaying the expression of the other 3 markers. The selected fields of view were exported to a JPG format. RESULTS: Assessing the co-expression of the antigens CD123, MNDA, CD68, and TCL1A detected 4 CD123+ cell subpopulations: No. 1. CD68+/ MNDA+/ TCL1A+; No. 2. CD68+/ MNDA+/ TCL1A-; No. 3. CD68+/ MNDA-/ TCL1A+; No. 4. CD68-/ MNDA-/ TCL1A+. CONCLUSION: SIMPLE has shown the phenotypic heterogeneity of CD123-positive cells (some of them may be PDCs) and could identify 4 immunophenotypically distinct subpopulations in the affected lymph nodes in patients with KFD. Further investigations are needed to define the role of subpopulations in the pathogenesis of KFD and other diseases.


Assuntos
Linfadenite Histiocítica Necrosante , Biópsia , Feminino , Linfadenite Histiocítica Necrosante/diagnóstico , Humanos , Subunidade alfa de Receptor de Interleucina-3 , Linfonodos , Coloração e Rotulagem
14.
Methods Mol Biol ; 2288: 217-232, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34270014

RESUMO

Here, we describe the first protocol of European radish (Raphanus sativus L. subsp. sativus convar. radicula) for obtaining doubled haploid plants through in vitro microspore culture, in which the full cycle of doubled haploid formation was successfully achieved. Using this protocol, a yield of up to eight embryoids per Petri dish can be obtained. Effectiveness of this protocol was confirmed for several genotypes of European radish.


Assuntos
Melhoramento Vegetal/métodos , Raphanus/crescimento & desenvolvimento , Raphanus/genética , Produtos Agrícolas/genética , Produtos Agrícolas/crescimento & desenvolvimento , Produtos Agrícolas/fisiologia , Meios de Cultura/química , Diploide , Corantes Fluorescentes , Genótipo , Haploidia , Homozigoto , Indóis , Biologia Molecular/métodos , Ploidias , Pólen/genética , Pólen/crescimento & desenvolvimento , Raphanus/fisiologia , Regeneração/genética , Coloração e Rotulagem , Técnicas de Cultura de Tecidos
15.
Methods Mol Biol ; 2288: 235-250, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34270015

RESUMO

Eggplant is one of the five important, worldwide-distributed solanaceous crops. The use of anther culture technology to produce pure, 100% homozygous doubled haploid lines for hybrid seed production is possible since 1982, where the first protocol of wide application to different eggplant materials was published. From then on, different improvements and adaptations to different materials have been made. In parallel, protocols to implement isolated microspore culture technology in eggplant have been developed principally in the last decade, which opens the door for a more efficient DH production in this species. In this chapter, two protocols, one for anther and other for isolated microspore culture in eggplant, are described. Some steps and materials are common to both approaches. A detailed description of each step from is provided.


Assuntos
Melhoramento Vegetal/métodos , Solanum melongena/crescimento & desenvolvimento , Solanum melongena/genética , Produtos Agrícolas/genética , Produtos Agrícolas/crescimento & desenvolvimento , Produtos Agrícolas/fisiologia , Meios de Cultura/química , Diploide , Flores/genética , Flores/crescimento & desenvolvimento , Corantes Fluorescentes , Haploidia , Homozigoto , Indóis , Biologia Molecular/métodos , Ploidias , Pólen/genética , Pólen/crescimento & desenvolvimento , Regeneração/genética , Solanum melongena/fisiologia , Coloração e Rotulagem , Técnicas de Cultura de Tecidos
16.
Methods Mol Biol ; 2288: 251-266, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34270016

RESUMO

The shed-microspore culture technique is an alternative sub-method combining anther and isolated microspore culture to induce microspore embryogenesis. Recently, its effective use in different types of peppers has drawn attention, because it has a higher embryo yield potential compared to anther culture and is more practical than isolated microspore culture. In this chapter, a stepwise protocol for shed-microspore culture of ornamental pepper is described. This protocol includes the steps of donor plant growth conditions, the choice of suitable flower buds based on DAPI staining of microspores, application of a cold pretreatment to flower buds, surface sterilization of the buds, shed-microspore culture of anthers, stress treatments, regeneration of androgenic in vitro plantlets, their acclimatization and ploidy analysis, and in vivo chromosome doubling of the haploid plants.


Assuntos
Capsicum/crescimento & desenvolvimento , Capsicum/genética , Melhoramento Vegetal/métodos , Pólen/crescimento & desenvolvimento , Pólen/genética , Capsicum/fisiologia , Produtos Agrícolas/genética , Produtos Agrícolas/crescimento & desenvolvimento , Produtos Agrícolas/fisiologia , Meios de Cultura/química , Diploide , Flores/genética , Flores/crescimento & desenvolvimento , Corantes Fluorescentes , Haploidia , Homozigoto , Indóis , Biologia Molecular/métodos , Ploidias , Regeneração/genética , Coloração e Rotulagem , Técnicas de Cultura de Tecidos
18.
Methods Mol Biol ; 2350: 191-227, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34331287

RESUMO

Fluorescence imaging has become a powerful tool for observations in biology. Yet it has also encountered limitations to overcome optical interferences of ambient light, autofluorescence, and spectrally interfering fluorophores. In this account, we first examine the current approaches which address these limitations. Then we more specifically report on Out-of-Phase Imaging after Optical Modulation (OPIOM), which has proved attractive for highly selective multiplexed fluorescence imaging even under adverse optical conditions. After exposing the OPIOM principle, we detail the protocols for successful OPIOM implementation.


Assuntos
Imunofluorescência/métodos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Imagem Óptica/métodos , Algoritmos , Animais , Corantes Fluorescentes , Processamento de Imagem Assistida por Computador , Luz , Modelos Teóricos , Coloração e Rotulagem
19.
Am J Pathol ; 191(8): 1431-1441, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34294192

RESUMO

Glomeruli instance segmentation from pathologic images is a fundamental step in the automatic analysis of renal biopsies. Glomerular histologic manifestations vary widely among diseases and cases, and several special staining methods are necessary for pathologic diagnosis. A robust model is needed to segment and classify glomeruli with different staining methods and apply in cases with various glomerular pathologic changes. Herein, pathologic images from renal biopsy slides stained with three basic special staining methods were used to build the data sets. The snapshot group included 1970 glomeruli from 516 patients, and the whole-slide image group included 8665 glomeruli from 148 patients. Cascade Mask region-based convolutional neural net architecture was trained to detect, classify, and segment glomeruli into three categories: i) GN, structural normal; ii) global sclerosis; and iii) glomerular with other lesions. In the snapshot group, total glomeruli, GN, global sclerosis, and glomerular with other lesions achieved an F1 score of 0.914, 0.896, 0.681, and 0.756, respectively, which were comparable with those in the whole-slide image group (0.940, 0.839, 0.806, and 0.753, respectively). Among the three categories, GN achieved the best instance segmentation effect in both groups, as determined by average precision, average recall, F1 score, and Mask mean Intersection over Union. The present model segments and classifies multistained glomeruli with efficiency and robustness. It can be applied as the first step for more detailed glomerular histologic analysis.


Assuntos
Aprendizado Profundo , Interpretação de Imagem Assistida por Computador/métodos , Nefropatias/diagnóstico , Glomérulos Renais/patologia , Biópsia , Humanos , Nefropatias/patologia , Coloração e Rotulagem
20.
Methods Mol Biol ; 2350: 267-287, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34331291

RESUMO

The UltraPlex method for multiplexed two-dimensional fluorescent immunohistochemistry is described, in which hapten tags conjugated to primary antibodies facilitate multiplexed imaging of four or more antigens per tissue section at once. Anti-hapten secondary antibodies labeled with fluorophores provide amplified signal for detection, which is accomplished using a standard fluorescent microscope or digital slide scanner. The protocol is rapid and straightforward and utilizes conventionally prepared tissue samples. The resulting staining is highly sensitive and specific, enabling high-resolution imaging of multiple cellular subtypes within tissue samples. Tumor cells and tumor-infiltrating lymphocytes are presented as examples. Multiple 4-plex-stained tissue samples can be digitally overlaid to create 8-plex (or more) high-content images, enabling visualization of distribution of complex cellular subtypes across tissues.


Assuntos
Imunofluorescência , Haptenos , Imuno-Histoquímica/métodos , Biomarcadores , Biomarcadores Tumorais , Análise de Dados , Humanos , Processamento de Imagem Assistida por Computador/métodos , Neoplasias/metabolismo , Neoplasias/patologia , Coloração e Rotulagem
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