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1.
Ann Lab Med ; 42(1): 47-53, 2022 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-34374348

RESUMO

Background: Since 2017, automated assays have been used in most clinical laboratories for anti-Müllerian hormone (AMH) level measurement. We evaluated the analytical performance of the newly developed automated fluorescent immunoassay system (AFIAS) AMH assay (Boditech Med, Gangwon-do, Korea) in comparison with the Roche Elecsys and Beckman Coulter Access 2 AMH assays. Methods: Analytical performance of the AFIAS AMH assay was assessed in terms of linearity, repeatability, and within-laboratory precision (CV%) using human recombinant AMH samples according to the Clinical and Laboratory Standards Institute (CLSI) guidelines EP05 and EP06. Using 293 serum samples collected from an infertility clinic, the AMH levels were compared across AFIAS, Elecsys, and Access 2 AMH assays according to the CLSI EP09 guidelines. Results: The AFIAS AMH assay results were linear across the measurement range of 0.420-72.386 pmol/L AMH, with repeatability of 6.341%. CV% of the AFIAS AMH assay for three levels of control, 1.786, 7.143, and 56.857 pmol/L, were 5.801%, 5.714%, and 6.228%, respectively. The results of the three AMH assays showed strong correlation: AFIAS and Elecsys [slope, 1.055 (95% confidence interval (CI), 1.022-1.088) and Spearman's rho, 0.978 (95% CI, 0.973-0.983)], Elecsys and Access 2 [slope, 0.813 (95% CI, 0.791-0.834) and Spearman's rho, 0.986 (95% CI, 0.983-0.989)], and AFIAS and Access 2 [slope, 0.836 (95% CI, 0.821-0.853) and Spearman's rho, 0.984 (95% CI, 0.980-0.988)]. Conclusions: The AFIAS AMH assay may be an alternative to the Roche Elecsys and Beckman Coulter Access 2 AMH assays.


Assuntos
Hormônio Antimülleriano , Serviços de Laboratório Clínico , Imunofluorescência , Humanos , Imunoensaio , Padrões de Referência
2.
Methods Mol Biol ; 2351: 307-320, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34382197

RESUMO

The transition from silenced heterochromatin to a biologically active state and vice versa is a fundamental part of the implementation of cell type-specific gene expression programs. To reveal structure-function relationships and dissect the underlying mechanisms, experiments that ectopically induce transcription are highly informative. In particular, the approach to perturb chromatin states by recruiting fusions of the catalytically inactive dCas9 protein in a sequence-specific manner to a locus of interest has been used in numerous applications. Here, we describe how this approach can be applied to activate pericentric heterochromatin (PCH) in mouse cells as a prototypic silenced state by providing protocols for the following workflow: (a) Recruitment of dCas9 fusion constructs with the strong transcriptional activator VPR to PCH. (b) Analysis of the resulting changes in chromatin compaction, epigenetic marks, and active transcription by fluorescence microscopy-based readouts. (c) Automated analysis of the resulting images with a set of scripts in the R programming language. Furthermore, we discuss how parameters for chromatin decondensation and active transcription are extracted from these experiments and can be combined with other readouts to gain insights into PCH activation.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Regulação da Expressão Gênica , Heterocromatina/genética , Ativação Transcricional , Animais , Proteína 9 Associada à CRISPR/genética , Cromatina/genética , Cromatina/metabolismo , Fibroblastos/metabolismo , Imunofluorescência/métodos , Expressão Gênica , Heterocromatina/metabolismo , Processamento de Imagem Assistida por Computador , Camundongos , Microscopia de Fluorescência , Ligação Proteica , Transfecção , Fluxo de Trabalho
3.
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
4.
Curr Protoc ; 1(7): e203, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34289261

RESUMO

The Drosophila melanogaster brain comprises different neuronal cell types that interconnect with precise patterns of synaptic connections. These patterns are essential for the normal function of the brain. To understand the connectivity patterns requires characterizing them at single-cell resolution, for which a fluorescence microscope becomes an indispensable tool. Additionally, because the neurons connect at the nanoscale, the investigation often demands super-resolution microscopy. Here, we adopt one super-resolution microscopy technique, called stochastic optical reconstruction microscopy (STORM), improving the lateral and axial resolution to ∼20 nm. This article extensively describes our methods along with considerations for sample preparation of neurons in vitro and in vivo, conjugation of dyes to antibodies, immunofluorescence labeling, and acquisition and processing of STORM data. With these tools and techniques, we open up the potential to investigate cell-cell interactions using STORM in the Drosophila nervous system. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Preparation of Drosophila primary neuronal culture and embryonic fillets Basic Protocol 2: Immunofluorescence labeling of samples Basic Protocol 3: Single-molecule fluorescence imaging Basic Protocol 4: Localization and visualization of single-molecule data Supporting Protocol: Conjugation of antibodies with STORM-compatible dyes.


Assuntos
Drosophila melanogaster , Drosophila , Animais , Imunofluorescência , Microscopia de Fluorescência , Neurônios
5.
Int J Mol Sci ; 22(12)2021 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-34204426

RESUMO

The last decade has witnessed the identification of several families affected by hereditary non-syndromic hearing loss (NSHL) caused by mutations in the SMPX gene and the loss of function has been suggested as the underlying mechanism. In the attempt to confirm this hypothesis we generated an Smpx-deficient zebrafish model, pointing out its crucial role in proper inner ear development. Indeed, a marked decrease in the number of kinocilia together with structural alterations of the stereocilia and the kinocilium itself in the hair cells of the inner ear were observed. We also report the impairment of the mechanotransduction by the hair cells, making SMPX a potential key player in the construction of the machinery necessary for sound detection. This wealth of evidence provides the first possible explanation for hearing loss in SMPX-mutated patients. Additionally, we observed a clear muscular phenotype consisting of the defective organization and functioning of muscle fibers, strongly suggesting a potential role for the protein in the development of muscle fibers. This piece of evidence highlights the need for more in-depth analyses in search for possible correlations between SMPX mutations and muscular disorders in humans, thus potentially turning this non-syndromic hearing loss-associated gene into the genetic cause of dysfunctions characterized by more than one symptom, making SMPX a novel syndromic gene.


Assuntos
Orelha Interna/embriologia , Orelha Interna/metabolismo , Proteínas Musculares/deficiência , Músculos/embriologia , Músculos/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Animais , Desenvolvimento Embrionário , Imunofluorescência , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Células Ciliadas Auditivas/metabolismo , Mecanotransdução Celular/genética , Desenvolvimento Muscular/genética , Organogênese/genética , Fenótipo , Transporte Proteico
7.
Int J Mol Sci ; 22(13)2021 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-34203300

RESUMO

Pain symptoms in temporomandibular disorders (TMD) predominantly affect reproductive women, suggesting that estrogen regulates pain perception. However, how estrogen contributes to chronic TMD pain remains largely unclear. In the present study, we performed behavioral tests, electrophysiology, Western blot and immunofluorescence to investigate the role and underlying mechanisms of estrogen in dental experimental occlusal interference (EOI)-induced chronic masseter mechanical hyperalgesia in rats. We found that long-term 17ß-estradiol (E2) replacement exacerbated EOI-induced masseter hyperalgesia in a dose-dependent manner in ovariectomized (OVX) rats. Whole-cell patch-clamp recordings demonstrated that E2 (100 nM) treatment enhanced the excitability of isolated trigeminal ganglion (TG) neurons in OVX and OVX EOI rats, and EOI increased the functional expression of transient receptor potential vanilloid-1 (TRPV1). In addition, E2 replacement upregulated the protein expression of TRPV1 in EOI-treated OVX rats. Importantly, intraganglionic administration of the TRPV1 antagonist AMG-9810 strongly attenuated the facilitatory effect of E2 on EOI-induced masseter mechanical sensitivity. These results demonstrate that E2 exacerbated EOI-induced chronic masseter mechanical hyperalgesia by increasing TG neuronal excitability and TRPV1 function. Our study helps to elucidate the E2 actions in chronic myogenic TMD pain and may provide new therapeutic targets for relieving estrogen-sensitive pain.


Assuntos
Hiperalgesia/tratamento farmacológico , Neurônios Aferentes/efeitos dos fármacos , Canais de Cátion TRPV/metabolismo , Gânglio Trigeminal/metabolismo , Acrilamidas/farmacologia , Animais , Western Blotting , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Estradiol/genética , Estradiol/metabolismo , Feminino , Imunofluorescência , Hiperalgesia/metabolismo , Ovariectomia , Ratos , Ratos Sprague-Dawley , Gânglio Trigeminal/efeitos dos fármacos
8.
Methods Mol Biol ; 2350: 43-68, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34331278

RESUMO

Förster resonance energy transfer (FRET) biosensors are popular and useful for directly observing cellular signaling pathways in living cells. Until recently, multiplex imaging of genetically encoded FRET biosensors to simultaneously monitor several protein activities in one cell was limited due to a lack of spectrally compatible FRET pair of fluorescent proteins. With the recent development of miRFP series of near-infrared (NIR) fluorescent proteins, we are now able to extend the spectrum of FRET biosensors beyond blue-green-yellow into NIR. These new NIR FRET biosensors enable direct multiplex imaging together with commonly used cyan-yellow FRET biosensors. We describe herein a method to produce cell lines harboring two compatible FRET biosensors. We will then discuss how to directly multiplex-image these FRET biosensors in living cells. The approaches described herein are generally applicable to any combinations of genetically encoded, ratiometric FRET biosensors utilizing the cyan-yellow and NIR fluorescence.


Assuntos
Técnicas Biossensoriais/métodos , Imunofluorescência/métodos , Transdução de Sinais , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Proteínas de Transporte , Linhagem Celular , Ativação Enzimática , Transferência Ressonante de Energia de Fluorescência/métodos , Genes Reporter , Camundongos , Ligação Proteica , Proteínas rho de Ligação ao GTP/genética
9.
Methods Mol Biol ; 2350: 125-144, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34331283

RESUMO

In this chapter, we describe the pipeline for multiplex immunohistochemical staining, multispectral image acquisition, and analysis. The protocol is dedicated for use on human formalin fixed paraffin embedded (FFPE) tissues and utilizes immune markers of dendritic cells, myeloid cells, and macrophages, as well as cytokeratin. This provides quantitative data of the (co-)expression levels and spatial localization of immune cell subtypes.


Assuntos
Imunofluorescência/métodos , Histocitoquímica/métodos , Biomarcadores , Análise de Dados , Formaldeído , Humanos , Processamento de Imagem Assistida por Computador , Inclusão em Parafina , Software , Fixação de Tecidos
10.
Methods Mol Biol ; 2350: 145-156, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34331284

RESUMO

Intravital two-photon microscopy enables monitoring of cellular dynamics and communication of complex systems, in genuine environment-the living organism. Particularly, its application in understanding the immune system brought unique insights into pathophysiologic processes in vivo. Here we present a method to achieve multiplexed dynamic intravital two-photon imaging by using a synergistic strategy combining a spectrally broad range of fluorophore emissions, a wave-mixing concept for simultaneous excitation of all targeted fluorophores, and an effective unmixing algorithm based on the calculation of spectral similarities with previously acquired fluorophore fingerprints. Our unmixing algorithm allows us to distinguish 7 fluorophore signals corresponding to various cellular and tissue compartments by using only four detector channels.


Assuntos
Imunofluorescência/métodos , Microscopia Intravital/métodos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Algoritmos , Animais , Linhagem Celular , Análise de Dados , Humanos , Processamento de Imagem Assistida por Computador , Microscopia Intravital/instrumentação , Camundongos , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação
11.
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
12.
Methods Mol Biol ; 2352: 117-126, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34324183

RESUMO

Somatic cell nuclear transfer and in vitro induction of pluripotency in somatic cells by defined factors provided unambiguous evidence that the epigenetic state of terminally differentiated somatic cells is not static and can be reversed to a more primitive one. Inspired by these results, stem cell biologists have identified approaches to directly convert fibroblasts into induced neuronal (iN) cells, indicating that direct lineage conversions are possible between distantly related cell types. More recently, we took advantages of pro-neurogenic capacity of iN factors and developed methods to rapidly derive functionally mature neurons directly from human pluripotent stem cells (hPSCs) through a brief induction of defined transcription factors. In this chapter, we describe the detailed methods used to attain the direct conversion from hPSCs to glutamatergic and GABAergic iN cells.


Assuntos
Diferenciação Celular , Neurônios/citologia , Neurônios/metabolismo , Células-Tronco Pluripotentes/citologia , Linhagem Celular , Separação Celular , Fibroblastos/citologia , Fibroblastos/metabolismo , Imunofluorescência , Neurônios GABAérgicos/citologia , Neurônios GABAérgicos/metabolismo , Vetores Genéticos/administração & dosagem , Vetores Genéticos/biossíntese , Vetores Genéticos/genética , Humanos , Lentivirus/genética , Neurogênese , Neuroglia/citologia , Neuroglia/metabolismo , Células-Tronco Pluripotentes/metabolismo , Fatores de Transcrição , Transdução Genética
13.
Am J Physiol Cell Physiol ; 321(3): C453-C470, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34260300

RESUMO

The myotendinous junction (MTJ) is a specialized interface for transmitting high forces between the muscle and tendon and yet the MTJ is a common site of strain injury with a high recurrence rate. The aim of this study was to identify previously unknown MTJ components in mature animals and humans. Samples were obtained from the superficial digital flexor (SDF) muscle-tendon interface of 20 horses, and the tissue was separated through a sequential cryosectioning approach into muscle, MTJ (muscle tissue enriched in myofiber tips attached to the tendon), and tendon fractions. RT-PCR was performed for genes known to be expressed in the three tissue fractions and t-distributed stochastic neighbor embedding (t-SNE) plots were used to select the muscle, MTJ, and tendon samples from five horses for RNA sequencing. The expression of previously known and unknown genes identified through RNA sequencing was studied by immunofluorescence on human hamstring MTJ tissue. The main finding was that RNA sequencing identified the expression of a panel of 61 genes enriched at the MTJ. Of these, 48 genes were novel for the MTJ and 13 genes had been reported to be associated with the MTJ in earlier studies. The expression of known [COL22A1 (collagen XXII), NCAM (neural cell adhesion molecule), POSTN (periostin), NES (nestin), OSTN (musclin/osteocrin)] and previously undescribed [MNS1 (meiosis-specific nuclear structural protein 1), and LCT (lactase)] MTJ genes was confirmed at the protein level by immunofluorescence on tissue sections of human MTJ. In conclusion, in muscle-tendon interface tissue enriched with myofiber tips, we identified the expression of previously unknown MTJ genes representing diverse biological processes, which may be important in the maintenance of the specialized MTJ.


Assuntos
Músculos Isquiossurais/metabolismo , Tendões dos Músculos Isquiotibiais/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/genética , RNA Mensageiro/genética , Adulto , Animais , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Colágeno/genética , Colágeno/metabolismo , Feminino , Imunofluorescência , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Ontologia Genética , Cavalos , Humanos , Masculino , Anotação de Sequência Molecular , Proteínas Musculares/classificação , Proteínas Musculares/metabolismo , Nestina/genética , Nestina/metabolismo , Moléculas de Adesão de Célula Nervosa/genética , Moléculas de Adesão de Célula Nervosa/metabolismo , RNA Mensageiro/classificação , RNA Mensageiro/metabolismo , Análise de Sequência de RNA , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
14.
Methods Mol Biol ; 2350: 77-93, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34331280

RESUMO

Multiplexed tissue tomography enables comprehensive spatial analysis of markers within a whole tissue or thick tissue section. Clearing agents are often used to make tissue transparent and facilitate deep tissue imaging. Many methods of clearing and tissue tomography are currently used in a variety of tissue types. Here we detail a workflow known as transparent tissue tomography (T3), which builds upon previous methods and can be applied to difficult to clear tissues such as tumors.


Assuntos
Imunofluorescência , Histocitoquímica/métodos , Imagem Óptica/métodos , Tomografia/métodos , Animais , Biomarcadores , Humanos , Imageamento Tridimensional/métodos , Camundongos , Especificidade de Órgãos , Fluxo de Trabalho
15.
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
16.
Methods Mol Biol ; 2350: 21-30, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34331276

RESUMO

Confocal immunofluorescence microscopy is an advanced imaging technique routinely applied in the laboratory and clinics. Histological analyses are performed from tissue material. In general, a single fluorochrome per laser is employed, limiting simultaneous analysis to four antigens in one staining with a conventional 4-laser line microscope. Here, we describe a protocol for combining fluorochromes with the same excitation but different emission properties that allows for the analysis of six different antigens in confocal immunofluorescence microscopy with a conventional 4-laser line microscope. The proposed multiplexed method permits the identification and characterization of complex cell populations in rare tissue material.


Assuntos
Imunofluorescência/métodos , Lasers , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Corantes Fluorescentes , Humanos , Microscopia Confocal/métodos
17.
Methods Mol Biol ; 2350: 95-104, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34331281

RESUMO

In multicellular organisms, most physiological and pathological processes involve an interplay between various cells and molecules that act both locally and systemically. To understand how these complex and dynamic processes occur in time and space, imaging techniques are key. Advances in tissue processing techniques and microscopy now allow us to probe these processes at a large scale and at the same time at a level of detail previously unachievable. Indeed, it is now possible to reliably quantify multiple protein expression levels at single-cell resolution in whole organs using three-dimensional fluorescence imaging techniques. Here we describe a method to prepare adult mouse bone tissue for multiplexed confocal imaging of thick tissue sections. Up to eight different fluorophores can be multiplexed using this technique and spectrally resolved using standard confocal microscopy. The optical clearing method described allows detection of these fluorophores up to a depth of >700 µm in the far-red. Although the method was initially developed for bone tissue imaging, we have successfully applied it to several other tissue types.


Assuntos
Imunofluorescência/métodos , Imageamento Tridimensional/métodos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Animais , Medula Óssea , Osso e Ossos , Camundongos , Especificidade de Órgãos
18.
Methods Mol Biol ; 2350: 105-123, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34331282

RESUMO

Early detection of malignant tumors, micrometastases, and disseminated tumor cells is one of the effective way of fighting cancer. Among the many existing imaging methods like computed tomography (CT), ultrasound (US), magnetic resonance imaging (MRI), positron emission tomography (PET), and single-photon emission computed tomography (SPECT), optical imaging with fluorescent probes is one of the most promising alternatives because it is fast, inexpensive, safe, sensitive, and specific. However, traditional fluorescent probes, based on organic fluorescent dyes, suffer from the low signal-to-noise ratio. Furthermore, conventional organic fluorescent dyes are unsuitable for deep tissue imaging because of the strong visible light absorption by biological tissues. The use of fluorescent semiconductor nanocrystals, or quantum dots (QDs), may overcome this limitation due to their large multiphoton cross section, which ensures efficient imaging of thick tissue sections inaccessible with conventional fluorescent probes. Moreover, the lower photobleaching and higher brightness of fluorescence signals from QDs ensures a much better discrimination of positive signals from the background. The use of fluorescent nanoprobes based on QDs conjugated to uniformly oriented high-affinity single-domain antibodies (sdAbs) may significantly increase the sensitivity and specificity due to better recognition of analytes and deeper penetration into tissues due to small size of such nanoprobes.Here, we describe a protocol for the fabrication of nanoprobes based on sdAbs and QDs, preparation of experimental xenograft mouse models for quality control, and multiphoton imaging of deep-tissue solid tumors, micrometastases, and disseminated tumor cells.


Assuntos
Imunofluorescência/métodos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Neoplasias/diagnóstico por imagem , Neoplasias/patologia , Pontos Quânticos , Anticorpos de Domínio Único , Linhagem Celular Tumoral , Imunofluorescência/normas , Humanos , Imunoconjugados/química , Imuno-Histoquímica/métodos , Sondas Moleculares , Imagem Multimodal/métodos , Nanopartículas , Micrometástase de Neoplasia , Imagem Óptica/métodos
19.
Methods Mol Biol ; 2350: 289-297, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34331292

RESUMO

Atomic force microscopy (AFM) enables the characterization of a wide range of samples including live cells. It is generally admitted that cancer cells are significantly softer than their normal counterparts, but imaging live cells by AFM using traditional modes can be at the cost of time or resolution. We describe how this tool can be used to estimate the motility of cancer versus normal cells, based on topographical and mechanical approaches, and coupled to optical imaging.


Assuntos
Movimento Celular , Microscopia de Força Atômica , Microscopia de Vídeo/métodos , Neoplasias/patologia , Imagem Óptica/métodos , Linhagem Celular Tumoral , Células Cultivadas , Imunofluorescência/métodos , Humanos , Microscopia de Força Atômica/métodos
20.
Methods Mol Biol ; 2319: 51-60, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34331242

RESUMO

Cardiovascular disease is a worldwide health issue that affects millions of lives every year, and thus, researchers are in need of high-throughput model systems with which to investigate mechanisms of disease and to develop and test potential therapies. The use of human-derived induced pluripotent stem cells (iPSCs) differentiated into cardiomyocytes aims to address this need. While cardiac differentiation protocols have been established previously in iPSCs, optimization of cardiac differentiation remains crucial to obtaining high quality cardiomyocytes for future experimental analyses. Important factors to consider include cell density and rate of proliferation, temporal regulation of media changes throughout the differentiation process, and the concentration of the chemicals utilized. In this chapter, we present a detailed protocol to outline the process of differentiating cardiomyocytes from human iPSCs via modulation of Wnt signaling, characterization of cardiomyocytes by immunofluorescence, as well as guidelines for troubleshooting and optimizing these techniques.


Assuntos
Técnicas de Cultura de Células/métodos , Meios de Cultura/química , Células-Tronco Pluripotentes Induzidas/citologia , Desenvolvimento Muscular , Miócitos Cardíacos/citologia , Via de Sinalização Wnt , Imunofluorescência , Humanos , Técnicas In Vitro , Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/metabolismo
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