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1.
Methods Mol Biol ; 2265: 141-154, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33704712

RESUMO

Three-dimensional (3D) cell culture has allowed a deeper understanding of complex pathological and physiological processes, overcoming some of the limitations of 2D cell culture on plastic and avoiding the costs and ethical issues related to experiments involving animals. Here we describe a protocol to embed single melanoma cells alone or together with primary human lymphatic endothelial cells in a 3D cross-linked matrix, to investigate the invasion and molecular crosstalk between these two cell types, respectively. After fixation and staining with antibodies and fluorescent conjugates, phenotypic changes in both cell types can be specifically analyzed by confocal microscopy.


Assuntos
Técnicas de Cocultura/métodos , Células Endoteliais/metabolismo , Melanoma/metabolismo , Esferoides Celulares/metabolismo , Linhagem Celular Tumoral , Células Endoteliais/citologia , Imunofluorescência/métodos , Humanos , Melanoma/patologia , Microscopia Confocal , Invasividade Neoplásica
2.
J Vis Exp ; (168)2021 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-33645557

RESUMO

Ambient fine particulate matter (PM2.5) exposure can lead to cardiac developmental toxicity but the underlying molecular mechanisms are still unclear. 8-hydroxy-2'deoxygenase (8-OHdG) is a marker of oxidative DNA damage and γH2AX is a sensitive marker for DNA double strand breaks. In this study, we aimed to detect PM2.5-induced 8-OHdG and γH2AX changes in the heart of zebrafish embryos using an immunofluorescence assay. Zebrafish embryos were treated with extractable organic matters (EOM) from PM2.5 at 5 µg/mL in the presence or absence of antioxidant N-acetyl-L-cysteine (NAC, 0.25 µM) at 2 h post fertilization (hpf). DMSO was used as a vehicle control. At 72 hpf, hearts were dissected from embryos using a syringe needle and fixed and permeabilized. After being blocked, samples were probed with primary antibodies against 8-OHdG and γH2AX. Samples were then washed and incubated with secondary antibodies. The resulting images were observed under fluorescence microscopy and quantified using ImageJ. The results show that EOM from PM2.5 significantly enhanced 8-OHdG and γH2AX signals in the heart of zebrafish embryos. However, NAC, acting as a reactive oxygen species (ROS) scavenger, partially counteracted the EOM-induced DNA damage. Here, we present an immunofluorescence protocol for investigating the role of DNA damage in PM2.5-induced heart defects that can be applied to the detection of environmental chemical-induced protein expression changes in the hearts of zebrafish embryos.


Assuntos
Dano ao DNA , Embrião não Mamífero/efeitos dos fármacos , Imunofluorescência/métodos , Coração/embriologia , Material Particulado/toxicidade , Peixe-Zebra/embriologia , Animais , Embrião não Mamífero/metabolismo , Coração/efeitos dos fármacos , Cardiopatias Congênitas/embriologia
3.
PLoS One ; 16(1): e0244601, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33497414

RESUMO

Several commercial Zika virus (ZIKV) serology assays have been developed since the recognition of ZIKV outbreaks as a Public Health Emergency of International Concern in 2016. However, test interpretation for ZIKV serology can be challenging due to antibody cross-reactivity with other flaviviruses like dengue virus (DENV). Therefore, we sought to evaluate the performance of eight commercially available ZIKV IgM and IgG assays across three testing platforms, namely, immunochromatographic tests (ICT), ELISAs and immunofluorescence tests (IIFT). The test panel comprised of 278 samples, including acute and convalescent sera or plasma from ZIKV-confirmed, DENV-confirmed, non-ZIKV and non-DENV patients, and residual sera from healthy blood donors. The ZIKV IgM and IgG serology assays yielded higher test sensitivities of 23.5% - 97.1% among ZIKV convalescent samples as compared to 5.6% - 27.8% among ZIKV acute samples; the test specificities were 63.3% - 100% among acute and convalescent DENV, non-DENV samples. Among the ELISAs and IIFTs, the Diapro ZIKV IgM ELISA demonstrated high test sensitivity (96%) and specificity (80%) when tested on early convalescent samples, while the Euroimmun ZIKV IgG ELISA yielded the highest test specificity of 97% - 100% on samples from non-ZIKV patients and healthy blood donors. For rapid ICTs, the LumiQuick IgM rapid ICT yielded low test sensitivity, suggesting its limited utility. We showed that commercial ZIKV IgM and IgG serology assays have differing test performances, with some having moderate to high test sensitivities and specificities when used in a dengue endemic setting, although there were limitations in IgG serology.


Assuntos
Imunoglobulina G/sangue , Imunoglobulina M/sangue , Infecção por Zika virus/sangue , Infecção por Zika virus/diagnóstico , Zika virus/isolamento & purificação , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Reações Cruzadas , Dengue/sangue , Dengue/diagnóstico , Dengue/imunologia , Vírus da Dengue/imunologia , Vírus da Dengue/isolamento & purificação , Ensaio de Imunoadsorção Enzimática/métodos , Imunofluorescência/métodos , Humanos , Imunoensaio/métodos , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Sensibilidade e Especificidade , Testes Sorológicos , Zika virus/imunologia , Infecção por Zika virus/imunologia
4.
Int J Mol Sci ; 22(2)2021 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-33440873

RESUMO

Parkinson's disease (PD) is a progressive neurodegenerative disease characterized by the loss of dopaminergic neurons in the substantia nigra. Several treatments for PD have focused on the management of physical symptoms using dopaminergic agents. However, these treatments induce various adverse effects, including hallucinations and cognitive impairment, owing to non-targeted brain delivery, while alleviating motor symptoms. Furthermore, these therapies are not considered ultimate cures owing to limited brain self-repair and regeneration abilities. In the present study, we aimed to investigate the therapeutic potential of human adipose-derived stem cells (hASCs) using magnetic nanoparticles in a 6-hydroxydopamine (6-OHDA)-induced PD mouse model. We used the Maestro imaging system and magnetic resonance imaging (MRI) for in vivo tracking after transplantation of magnetic nanoparticle-loaded hASCs to the PD mouse model. The Maestro imaging system revealed strong hASCs signals in the brains of PD model mice. In particular, MRI revealed hASCs distribution in the substantia nigra of hASCs-injected PD mice. Behavioral evaluations, including apomorphine-induced rotation and rotarod performance, were significantly recovered in hASCs-injected 6-OHDA induced PD mice when compared with saline-treated counterparts. Herein, we investigated whether hASCs transplantation using magnetic nanoparticles recovered motor functions through targeted brain distribution in a 6-OHDA induced PD mice. These results indicate that magnetic nanoparticle-based hASCs transplantation could be a potential therapeutic strategy in PD.


Assuntos
Tecido Adiposo/citologia , Nanopartículas de Magnetita , Doença de Parkinson/terapia , Transplante de Células-Tronco , Células-Tronco/citologia , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Terapia Baseada em Transplante de Células e Tecidos/efeitos adversos , Terapia Baseada em Transplante de Células e Tecidos/métodos , Modelos Animais de Doenças , Imunofluorescência/métodos , Humanos , Imuno-Histoquímica , Camundongos , Oxidopamina/efeitos adversos , Doença de Parkinson/diagnóstico , Doença de Parkinson/etiologia , Transplante de Células-Tronco/efeitos adversos , Transplante de Células-Tronco/métodos , Células-Tronco/metabolismo
5.
Cytometry B Clin Cytom ; 100(1): 33-41, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33394568

RESUMO

Over a remarkably short period of time, a great deal of knowledge about severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) infection has been acquired, through the focused and cooperative effort of the international scientific community. Much has become known about how the immune response is coordinated to fight infection, and how it becomes dysregulated in severe disease. In this review, we take an in-depth look at the many immune features associated with the host response to SARS-CoV2, as well as those that appear to mark severe disease.


Assuntos
/diagnóstico por imagem , Citometria de Fluxo/métodos , Imunofluorescência/métodos , /imunologia , Biomarcadores/análise , /terapia , Quimiocinas/análise , Quimiocinas/metabolismo , Citocinas/análise , Citocinas/metabolismo , Imunofluorescência/tendências , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade/fisiologia , Metabolômica/métodos , Metabolômica/tendências , Medição de Risco , Índice de Gravidade de Doença
6.
Arch Virol ; 166(3): 715-731, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33492524

RESUMO

Coronaviruses (CoV) are a family of viral pathogens that infect both birds and mammals, including humans. Seven human coronaviruses (HCoV) have been recognized so far. HCoV-229E, -OC43, -NL63, and -HKU1 account for one-third of common colds with mild symptoms. The other three members are severe acute respiratory syndrome (SARS)-CoV, Middle East respiratory syndrome (MERS)-CoV, and SARS-CoV-2. These viruses are responsible for SARS, MERS, and CoV disease 2019 (COVID-19), respectively. A variety of diagnostic techniques, including chest X-rays, computer tomography (CT) scans, analysis of viral nucleic acids, proteins, or whole virions, and host antibody detection using serological assays have been developed for the detection of these viruses. In this review, we discuss conventional serological tests, such as enzyme-linked immunosorbent assay (ELISA), western blot (WB), immunofluorescence assay (IFA), lateral flow immunoassay (LFIA), and chemiluminescence immunoassay (CLIA), as well as biosensor-based assays that have been developed for diagnosing HCoV-associated diseases since 2003, with an in-depth focus on COVID-19.


Assuntos
Anticorpos Antivirais/sangue , /diagnóstico , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Síndrome Respiratória Aguda Grave/diagnóstico , Anticorpos Antivirais/imunologia , Técnicas Biossensoriais/métodos , Western Blotting/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Imunofluorescência/métodos , Humanos , Medições Luminescentes/métodos , Vírus da SARS/imunologia , Síndrome Respiratória Aguda Grave/virologia
7.
Methods Mol Biol ; 2230: 337-344, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33197023

RESUMO

Immunohistochemistry, or immunolabeling, is a key method for the identification of protein expression and localization. Successful detection relies on a low signal-to-noise ratio, which is affected greatly by antibody specificity as well as the staining protocol. Immunohistochemistry in the mouse is challenging, particularly in adult skeletal tissue, due to the need for long decalcification, high autofluorescence and high levels of endogenous peroxidase. Here, we describe a highly sensitive protocol for protein detection in decalcified paraffin-embedded sections from adult mouse skeletal tissue. By using four levels of amplification, this method allows for the identification of even low-abundance proteins.


Assuntos
Osso e Ossos/ultraestrutura , Técnica de Descalcificação/métodos , Imunofluorescência/métodos , Proteínas/isolamento & purificação , Coloração e Rotulagem/métodos , Animais , Osso e Ossos/diagnóstico por imagem , Humanos , Camundongos , Inclusão em Parafina/métodos , Proteínas/química
8.
Methods Mol Biol ; 2237: 1-10, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33237404

RESUMO

Sandwich-based antibody arrays enable the detection of multiple proteins simultaneously, thus offering a time- and cost-effective alternative to single-plex platforms. The protein of interest is "sandwiched" between an antibody that captures it to the array and a second antibody that is used for detection. Here we describe a 1-day procedure to process samples, such as serum or cell lysates, with a quantitative sandwich-based antibody array on a glass substrate using fluorescence.


Assuntos
Imunofluorescência/métodos , Testes Imunológicos/métodos , Análise Serial de Proteínas/métodos , Animais , Anticorpos/imunologia , Citocinas/análise , Citocinas/imunologia , Humanos
9.
Methods Mol Biol ; 2237: 39-44, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33237406

RESUMO

The sandwich-based immunoassay is renowned for its specificity and sensitivity for protein detection, where the antigen is "sandwiched" by a pair of antigen epitope-specific antibodies. The capture antibody-antigen-detection antibody complex will be formed if all the components are present in a proper reaction system. A one-step rapid sandwich-based antibody array can be developed through fixing the capture antibody on a glass slide with a fluorescence-labelled detection antibody. In this chapter, we describe the process of a one-step mouse immunoglobulin isotyping array for use in hybridoma culture supernatants.


Assuntos
Testes Imunológicos/métodos , Análise Serial de Proteínas/métodos , Animais , Imunofluorescência/métodos , Humanos , Imunoglobulinas/imunologia
10.
Methods Mol Biol ; 2237: 45-53, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33237407

RESUMO

Because of narrow availability of antibody pairs and potential cross-reactivity between antibodies, the development of sandwich-based antibody arrays which need a pair of antibodies for each target has been restricted to higher density resulting in limited proteomic breadth of detection. Label-based array is one way to overcome this obstacle by directly labeling all targets in samples with fluorescent dyes such as Cy3 and Cy5. The labeled samples are then applied on the antibody array chip composed of capture antibodies. In this chapter, we will introduce this technology including array production and sample detection assay.


Assuntos
Imunofluorescência/métodos , Análise Serial de Proteínas/métodos , Proteômica/métodos , Animais , Biotinilação/métodos , Corantes Fluorescentes/química , Humanos , Imunoensaio/métodos , Proteoma/química , Proteoma/imunologia
11.
Methods Mol Biol ; 2198: 217-226, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32822035

RESUMO

Immunostaining (also called as immunofluorescence) is a fluorescence labeling method to stain one or more epitopes of interest on DNA and/or protein using specific antibodies. Cytosine modifications can be detected quantitatively by immunostaining. The protocol commonly includes sequential steps. These include fixation, permeabilization, antigen retrieval, blocking, incubation with primary and secondary antibodies, and visualization under the microscope followed by image-based intensity analysis of staining. Each step is important, but antigen retrieval is especially necessary for DNA epitopes such as cytosine modifications as antibodies can access cytosines in DNA only once the DNA double-strand is denatured and DNA-packaging proteins have been removed. Hydrochloric acid is commonly used for this purpose. However, there are additional treatments with enzymes to enhance antigen retrieval and improve the detection by increasing staining intensity. This chapter describes current methodology for improving antigen retrieval for the staining of the cytosine modifications 5'-methylcytosine (5meC), 5'-hydroxymethylcytosine (5hmC), 5'-formylcytosine (5fC), and 5'-carboxycytosine (5caC).


Assuntos
Citosina/imunologia , Metilação de DNA/imunologia , Imunofluorescência/métodos , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/química , Animais , Anticorpos/imunologia , Antígenos/metabolismo , Citosina/análogos & derivados , Citosina/química , Citosina/metabolismo , DNA/metabolismo , Metilação de DNA/genética , Epigênese Genética/genética , Humanos
12.
Methods Mol Biol ; 2198: 255-268, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32822037

RESUMO

Male infertility is associated with several causes affecting the paternal nucleus such as DNA lesions (breaks, deletions, mutations, ...) or numerical chromosome anomalies. More recently, male infertility has also been associated with changes in the sperm epigenome, including modification in the topology of chromatin (Olszewska et al., Chromosome Research 16:875-890, 2008; Alladin et al., Syst Biol Reprod Med 59: 146-152, 2013) ref with number 1, 2. Indeed, the positioning of chromosomes in the sperm nucleus is nonrandom and defines chromosome territories (Champroux et al., Genes (Basel) 9:501, 2018) ref with number 3 whose optimal organization determines the success of embryonic development. In this context, the study of the spatial distribution of chromosomes in sperm cells could be relevant for clinical diagnosis. We describe here a in situ fluorescence hybridization (FISH) strategy coupled with a fluorescent immunocytochemistry approach followed by confocal analysis and reconstruction (2D/3D) as a powerful tool to analyze the location of chromosomes in the sperm nucleus using the mouse sperm as a model. Already, the two-dimensional (2D) analysis of FISH and immunofluorescence data reveal the location of chromosomes as well as the different markings on the spermatic nucleus. In addition, a good 3D rendering after Imaris software processing was obtained when Z-stacks of images were acquired over a defined volume (10 µm × 13 µm × 15 µm) with a sequential scanning mode to minimize bleed-through effects and avoid overlapping wavelengths.


Assuntos
Posicionamento Cromossômico/imunologia , Microscopia Confocal/métodos , Espermatozoides/imunologia , Aneuploidia , Animais , Núcleo Celular/imunologia , Cromatina , Aberrações Cromossômicas , Posicionamento Cromossômico/genética , Cromossomos/imunologia , Modelos Animais de Doenças , Imunofluorescência/métodos , Hibridização in Situ Fluorescente/métodos , Infertilidade Masculina/imunologia , Masculino , Camundongos , Espermatozoides/citologia
13.
Methods Mol Biol ; 2187: 327-335, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32770516

RESUMO

Fluorescence microscopy is a powerful and widely used tool in molecular biology. Over the years, the discovery and development of lipid-binding fluorescent probes has established new research possibilities to investigate lipid composition and dynamics in the cell. For instance, fluorescence microscopy has allowed the investigation of lipid localization and density in specific cell compartments such as membranes or organelles. Often, the characteristics and the composition of lipid-enriched structures are determined by analyzing the distribution of a fluorescently labeled lipid probe, which intercalates in lipid-enriched platforms, or specifically binds to parts of the lipid molecule. However, in many cases antibodies targeting proteins have higher specificity and are easier to generate. Therefore, we propose to use both antibodies targeting lipid transporters and lipid binding probes to better monitor lipid membrane changes. As an example, we visualize lipid rafts using the fluorescently labeled-B-subunit of the cholera toxin in combination with antibodies targeting ceramide-binding proteins CERTs, central molecules in the metabolism of sphingolipids.


Assuntos
Proteínas de Transporte/metabolismo , Imunofluorescência/métodos , Lipídeos de Membrana/metabolismo , Microdomínios da Membrana/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Anticorpos/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Toxina da Cólera/metabolismo , Corantes Fluorescentes/metabolismo , Células HEK293 , Humanos , Proteínas de Membrana/metabolismo , Esfingolipídeos/metabolismo
14.
Nat Biomed Eng ; 5(1): 53-63, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33349659

RESUMO

Biosensors that continuously measure circulating biomolecules in real time could provide insights into the health status of patients and their response to therapeutics. But biosensors for the continuous real-time monitoring of analytes in vivo have only reached nanomolar sensitivity and can measure only a handful of molecules, such as glucose and blood oxygen. Here we show that multiple analytes can be continuously and simultaneously measured with picomolar sensitivity and sub-second resolution via the integration of aptamers and antibodies into a bead-based fluorescence sandwich immunoassay implemented in a custom microfluidic chip. After an incubation time of 30 s, bead fluorescence is measured using a high-speed camera under spatially multiplexed two-colour laser illumination. We used the assay for continuous quantification of glucose and insulin concentrations in the blood of live diabetic rats to resolve inter-animal differences in the pharmacokinetic response to insulin as well as discriminate pharmacokinetic profiles from different insulin formulations. The assay can be readily modified to continuously and simultaneously measure other blood analytes in vivo.


Assuntos
Glicemia/análise , Imunofluorescência/métodos , Insulina/sangue , Técnicas Analíticas Microfluídicas/instrumentação , Animais , Diabetes Mellitus Experimental , Desenho de Equipamento , Imunofluorescência/instrumentação , Masculino , Ratos , Ratos Sprague-Dawley
15.
Methods Mol Biol ; 2221: 261-273, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32979208

RESUMO

Immunostaining is the process of identifying proteins in tissue sections by incubating the sample with antibodies specific to the protein of interest, then visualizing the bound antibody using a chromogen (immunohistochemistry or IHC) or fluorescence (immunofluorescence or IF). Unlike in situ hybridization, which identifies gene transcripts in cells, immunostaining identifies the products themselves and provides information about their localization within cells (nuclear, cytoplasmic, or membrane) or extracellular matrix. This can be particularly important in the context of bone and cartilage because they contain many cell types as well as matrix components, each with distinct protein expression patterns. As the number of antibodies continues to grow, this technique has become vital for research laboratories studying the skeleton. Here, we describe a detailed protocol for antibody-based in situ analysis of bone and associated tissues, addressing specific issues associated with staining of hard and matrix-rich tissues.


Assuntos
Osso e Ossos/química , Cartilagem/química , Imunofluorescência/métodos , Imuno-Histoquímica/métodos , Proteínas/análise , Fixação de Tecidos/métodos , Animais , Anticorpos/química , Humanos , Coloração e Rotulagem
16.
Methods Mol Biol ; 2220: 201-215, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32975777

RESUMO

The pathogen Listeria monocytogenes is a facultative intracellular bacterium, which targets a large range of cell types. Following entry, bacteria disrupt the invasion vacuole and reach the cytoplasm where they replicate and use the actin cytoskeleton to propel themselves from cell to cell. Mammalian epithelial cells grown in vitro can be used to study the different steps of the intracellular life of Listeria. However, rapid multiplication and dissemination of bacteria can induce important cell death and detachment, resulting in the formation of lytic plaques. Thus, in vitro infections with L. monocytogenes are usually restricted to short time courses, from a few minutes to one day. Here, we present a method to study long-term L. monocytogenes infections in epithelial cells using epifluorescence microscopy. This protocol enables the observation of actin-based motility, intercellular dissemination foci, and entrapment of L. monocytogenes within vacuoles of persistence termed "Listeria-Containing Vacuoles" (LisCVs). We also describe a protocol to study the recruitment of cytoskeletal proteins at Listeria actin comet tails, as well as a method to assess the membrane integrity of intracellular bacteria using a LIVE/DEAD viability assay.


Assuntos
Células Epiteliais/microbiologia , Listeria monocytogenes/fisiologia , Listeriose/patologia , Microscopia de Fluorescência/métodos , Linhagem Celular , Proteínas do Citoesqueleto/análise , Células Epiteliais/patologia , Imunofluorescência/métodos , Interações Hospedeiro-Patógeno , Humanos , Listeria monocytogenes/isolamento & purificação , Listeriose/microbiologia
17.
Methods Mol Biol ; 2219: 81-97, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33074535

RESUMO

To better understand the origin of animal cell types, body plans, and other morphological features, further biological knowledge and understanding are needed from non-bilaterian phyla, namely, Placozoa, Ctenophora, and Porifera. This chapter describes recent cell staining approaches that have been developed in three phylogenetically distinct sponge species-the homoscleromorph Oscarella lobularis, and the demosponges Amphimedon queenslandica and Lycopodina hypogea-to enable analyses of cell death, proliferation, and migration. These methods allow for a more detailed understanding of cellular behaviors and fates, and morphogenetic processes in poriferans, building on current knowledge of sponge cell biology that relies chiefly on classical (static) histological observations.


Assuntos
Poríferos/citologia , Coloração e Rotulagem/métodos , Animais , Rastreamento de Células/métodos , Imunofluorescência/métodos , Imagem Óptica/métodos
18.
Methods Mol Biol ; 2245: 215-224, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33315205

RESUMO

Immunofluorescence staining is a widely used and powerful tool for the visualization and colocalization of two or more proteins and/or cellular organelles. For colocalization studies in fixed cells, one target protein/organelle is immunostained and visualized by one fluorophore and the other target protein/organelle is immunostained and visualized by a different fluorophore whose excitation emission spectra does not overlap with the first fluorophore. Parkin (PARK2) is an E3 ubiquitin ligase which performs ubiquitination of surface proteins of dysfunctional mitochondria to mark them for autolysosomal degradation. Here we describe the immunofluorescence staining of parkin protein and immunofluorescence or dye-based methods to visualize mitochondria and study the colocalization of parkin and mitochondria in primary human or mouse chondrocytes or cell lines.


Assuntos
Condrócitos/metabolismo , Imunofluorescência , Mitocôndrias/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Biomarcadores , Células Cultivadas , Imunofluorescência/métodos , Humanos , Microscopia Confocal/métodos , Mitocôndrias/genética , Osteoartrite/genética , Osteoartrite/metabolismo , Osteoartrite/patologia , Permeabilidade , Transporte Proteico , Ubiquitina-Proteína Ligases/genética
19.
Methods Mol Biol ; 2214: 1-10, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32944899

RESUMO

A couple of days after fertilization of a mouse oocyte by a sperm, two sequential cell differentiation events segregate pluripotent cells that can be identified by the presence of specific markers. Early mammalian embryos are relatively easy to recover as they are not yet implanted in the uterus matrix. Several decades of experimentation have enabled to find appropriate media to culture them, and therefore provide an excellent way to test different experimental setups such as the use of signaling inhibitors. We provide here a commonly used protocol to culture preimplantation embryos as well as a method to detect pluripotent cells in blastocysts.


Assuntos
Blastocisto/citologia , Técnicas de Cultura Embrionária/métodos , Camundongos/embriologia , Células-Tronco Pluripotentes/citologia , Animais , Embrião de Mamíferos/citologia , Imunofluorescência/métodos , Microscopia de Fluorescência/métodos
20.
Methods Mol Biol ; 2214: 143-155, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32944908

RESUMO

Immunofluorescence staining enables the visualization of protein expression at a cellular or even sub-nuclear level. Whole-mount staining preserves the three-dimensional spatial information in biological samples allowing a comprehensive interpretation of expression domains. Here we describe the sample processing, protein detection using antibodies and confocal imaging of isolated preimplantation to early postimplantation mouse embryos up to Embryonic day 8.0 (E8.0).


Assuntos
Embrião de Mamíferos/ultraestrutura , Imunofluorescência/métodos , Camundongos/embriologia , Microscopia Confocal/métodos , Animais , Embrião de Mamíferos/embriologia , Microscopia de Fluorescência/métodos
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