A pesquisa descrita neste estudo foi apoiado por uma bolsa da National Science Foundation (DEB-0805818) para WAF, AWS e ID Este estudo foi também apoiado por doações IOS-0845847 a AWS, IOS-0919855 para AWS e ID e IOS-0920720 para WAF

<html> 1. Criação e manipulação da dieta para produzir variação no tamanho do corpo e da asa tamanho Racional e Visão. A expressão das relações de escala em última análise, depende da regulamentação processos de desenvolvimento que produzem variação no órgão final e tamanho do corpo. Estes processos são melhor descritos no trabalho de insetos totalmente metamórfica (ie, holometabolous), como D. melanogaster, onde órgãos crescer como indiferenciado &quot;discos imaginais&quot; dentro do grub-como larva 11,15-17. Em Drosophila, órgão final e tamanho do corpo são regulados pelo crescimento durante as fases larval; larvas crescem através do primeiro e segundo instar larval até atingirem um peso mínimo viável para a eclosão (MVW E) para o início do terceiro instar larval 18. Fome antes da realização MVW E impede a eclosão dos adultos. No entanto, as larvas que estão sedentos de MVW E gerar adultos viáveis, embora a um tamanho muito pequeno. Isto é porque reduziu a nutrição durante o desenvolvimento corporal final e reduz tamanho do órgão, e não apenas em moscas, mas em quase todos os animais 19-21. Ao manipular a disponibilidade de alimentos após MVW E é atingido, mas antes de pupação ocorre, é possível induzir extrema plasticidade fenotípica no tamanho do corpo que supera em muito outras fontes de variação (por exemplo, a variação genética individual). <ol><li> Os ovos são recolhidos junto da população a ser fenotipados, dividido em lotes de 50 e colocados em frascos contendo 10 ml de alimento voar padrão. Porque oviposição ocorre continuamente, os ovos são coletados a cada 24 horas durante três dias, produzindo três coortes de idade. </li><li> Quando o mais velho de coorte atinge o estágio errantes ou está apenas começando a pupate, a manipulação da dieta é aplicada. Neste ponto, a coorte mais velha é muito além do MVW E, o mais jovem de coorte é muito próximo do E MVW e da coorte restantes está em algum lugar entre esses extremos. Assim, a supressão dos alimentos neste momento irá produzir adultos muito grande a partir da coorte mais velha, adultos muito pequena da coorte mais jovem e de tamanho intermediário adultos da coorte de remanescentes. Para remover as larvas dos frascos, aproximadamente 5 ml de solução de sacarose 40% é adicionado a todos os frascos. Para liberar as larvas, a comida é agitada por 15-20 minutos em uma mesa shaker definida para uma velocidade intermediária. </li><li> Larvas flutuantes são removidos da solução de sacarose com um pincel (00) fina, colocados em frascos contendo um tampão de algodão molhado e permitiu a pupate. Alternativamente, as larvas podem ser colocados em um aditivo alimentar não digeríveis, como metil-celulose 22. </li></ol> 2. Estimativa do tamanho do corpo Racional e Visão. Estudos anteriores indicam que o comprimento do tórax é um menos de procuração ideal para o tamanho total do corpo 22. Em vez disso, usamos tamanho pupal como uma medida de tamanho do corpo. Tamanho corporal máximo é fixado a cessação da alimentação que antecede a pupação. Como resultado, há uma estreita correlação entre o tamanho da pupa e adulto tamanho corpo 23. <ol><li> Pupas são dispostas lado ventral para cima em lâminas de vidro para microscópio e fotografada usando uma câmera digital acoplada a um microscópio de dissecação. Cada imagem é capturada, atribuído um código de identificação único e transferidos para um computador através de uma transmissão ao vivo de conectar a câmera digital para o computador. </li><li> Pupas com imagens são movidas para cada 2 ml contendo 1ml epitubes de alimentos voar, identificado com o código único para cada indivíduo, e puncionada com respiradouro para permitir a troca de gás. </li><li> Tamanho pupal é estimado como o número de pixels da silhueta pupal. Métricas de outro tamanho também pode ser usado. </li></ol> 3. Traço estimativa do tamanho (asa) Racional e Visão. Para asas de imagem, usamos uma &#39;asa grabber&#39; 24, que puxa e prende a asa de um viver perpendicular voar, anestesiados ao seu corpo. Pressionado entre dois pedaços de vidro, isto permite que a asa a ser trabalhada como um objeto bidimensional. <ol><li> Moscas são anestesiados levemente com CO 2 e colocado em posição no grabber asa em um microscópio de dissecação equipado com uma câmera digital que é conectado a um computador. </li><li> A ala é fotografada e guardada sob o código único para cada indivíduo. </li><li> Tamanho da asa pode ser estimada utilizando o polígono convexo mínimo que circunscreve marginal marcos fixo ou como a contagem de pixels da silhueta da asa. </li></ol> 4. Estimativa de relação de escala <ol><li> Os dados são partilhados com outras gerações, mas separadas por sexo e log-transformada. </li><li> Relações de escala estão aptos para os dados de tamanho usando Tipo II (ou seja, redução do Eixo Maior) de regressão (ver revisão em 12) e os parâmetros estimados para cada grupo. </li></ol> 5. Resultados representativos: tenda &quot;&gt; Nossa manipulação da dieta irá produzir uma ampla gama de tamanhos de corpo (Figura 1) e tamanhos traço (Figura 2). Plotagem os traços log transformados uns contra os outros revela seu padrão de covariação dentro de um determinado grupo biológico e permite ajustar a relação de escala para o grupo (Figura 3). extração dos parâmetros relação de escala que descrevem como escalas forma com o tamanho do corpo permite a comparação de covaries como a forma com tamanho entre os grupos. <img alt="Figura 1" src="/files/ftp_upload/3162/3162fig1.jpg" /> Figura 1. Pupa dos extremos da distribuição de tamanho produzido através de manipulação de dieta após indivíduos alcançar o E MVW. Imagens reais pupal são mostrados à esquerda com suas silhuetas correspondente para a medição do tamanho pupal à direita. Barra de escala é de 1mm. <img alt="Figura 2" src="/files/ftp_upload/3162/3162fig2.jpg" /> Asas figura 2. Tirado extremos da distribuição de tamanho corporal produzida por meio de manipulação da dieta após indivíduos alcançar o E MVW. Imagens ala real são mostrados à esquerda com suas silhuetas correspondente para a medição do tamanho da asa mostrado à direita. Barra de escala é de 1mm. <img alt="Figura 3" src="/files/ftp_upload/3162/3162fig3.jpg" /> Figura 3. Asa-corpo de escala de tamanho de Drosophila melanogaster por sexo. Dados log-transformados para os indivíduos de cada sexo, que foram totalmente alimentado (círculos escuros), uma fome (círculos cinza) ou dois dias (círculos abertos), como larvas. Tipo de linha de regressão II é mostrado para cada sexo. Entre cada faixa etária, o declínio na área de asa média de 0,1 milímetros ~ (~ 7%) e ~ 0,2 milímetros (~ 8%) ocorre, aumentando a gama total de fenótipos observados. As regressões revelam que as fêmeas são ligeiramente hypoallometric para esta relação de escala (inclinação de 0,96), enquanto os machos são ligeiramente hyperallometric (inclinação 1,06).

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Escala morfológica tem sido objeto de intenso estudo em biologia evolutiva por quase um século. Mais recentemente, a base próxima da regulação do crescimento e ampliação tornou-se cada vez mais importante no desenvolvimento, fisiologia e genética (por exemplo, visualizar em 12,15,23-25). Nossa metodologia permite estimar relações de escala morfológica em toda a gama de traço e tamanhos de corpo. Isso pode ser importante, como o tamanho do traço pode não escala linearmente com o tamanho do corpo (por exemplo, 13,14). No entanto, isso pode não ser aparente se os animais são criados somente sob condições de laboratório ideal. Embora o nosso protocolo foi desenvolvido explicitamente para permitir uma estimativa da relação de escala de asa-corpo tamanho em Drosophila, ele pode ser usado com o traço específico de modificação para estimar a relação de escala para qualquer característica morfológica em moscas. Da mesma forma, a manipulação da dieta pode provavelmente ser usado para produzir toda a gama de tamanhos de traço e corpo em outros insetos totalmente metamórfica; aplicações como simplesmente exigir saber quando o MVW E é atingida e aplicar a manipulação nesse ponto na ontogenia.

experimental manipulation of body size to estimate morphological scaling relationships in drosophila

O dimensionamento de partes do corpo é uma característica central da morfologia animais 1-7. Dentro da espécie, características morfológicas precisam ser corretamente proporcionada ao corpo para o organismo para funcionar; indivíduos maiores normalmente têm partes maiores do corpo e indivíduos menores geralmente têm partes menores do corpo, de modo que a forma do corpo em geral é mantida através de uma variedade de tamanhos de corpo adulto. A exigência de proporções corretas significa que os indivíduos dentro das espécies geralmente apresentam baixa variação no tamanho característica relativa. Em contraste, tamanho característica relativa pode variar drasticamente entre as espécies e é um mecanismo primário pelo qual a diversidade morfológica é produzido. Mais de um século de trabalho comparativo estabeleceu essas intra e interespecífica padrões de 3,4. Talvez a abordagem mais amplamente usado para descrever esta variação é calcular a relação de escala entre o tamanho de dois traços morfológicos, utilizando a equação alométrica y = bxα, onde x e y são do tamanho de dois traços, como órgão e tamanho do corpo 8 , 9. Esta equação descreve a relação intra-grupo de escala (por exemplo, espécies população,) entre dois traços que ambos variam em tamanho. Log-transformação desta equação produz uma simples equação linear, log (y) = log (b) + αlog (x) e log-log parcelas do tamanho de diferentes características entre os indivíduos da mesma espécie tipicamente revelam escala linear com intercepto de log (b) e uma inclinação de α, o chamado &quot;coeficiente alométrico &#39;9,10. Variação morfológica entre os grupos é descrito por diferenças na escala intercepta relacionamento ou pistas para um par de determinado traço. Conseqüentemente, a variação nos parâmetros da equação alométrica (b e α) elegantemente descreve a variação forma capturado no relacionamento entre órgãos e tamanho do corpo dentro e entre grupos biológicos (ver 11,12). Nem todos os traços de escala linearmente com o outro ou com o tamanho do corpo (por exemplo, 13,14) Assim, as relações de escala morfológicas são mais informativos quando os dados são retirados da vasta gama de tamanhos de traço. Aqui nós descrevemos como simples manipulação experimental da dieta pode ser usado para produzir toda a gama de tamanho do corpo dos insetos. Isto permite uma estimativa da relação de escala total para um dado par de características, permitindo uma descrição completa de covaries como a forma com o tamanho e uma comparação robusto de parâmetros de escala do relacionamento entre os grupos biológicos. Embora nos concentremos em Drosophila, nossa metodologia deve ser aplicado a quase qualquer inseto totalmente metamórfica.

Watch this Scientific Journal Video about Experimental Manipulation of Body Size to Estimate Morphological Scaling Relationships in Drosophila at JoVE.com

Experimental Manipulation of Body Size to Estimate Morphological Scaling Relationships in Drosophila

Relações de escala morfológica captar e descrever a forma do organismo. Nós apresentamos um método para medir as relações de escala morfológica em toda a gama natural de tamanhos de corpo dos insetos totalmente metamórfica. Usando uma simples manipulação da dieta que aumentar a distribuição de tamanhos de traço, permitindo a descrição exata de como a forma e tamanho co-variar.

A manipulação experimental de tamanho do corpo para Estimar Relacionamentos Scaling morfológicas em Drosophila</em

The scaling of body parts is a central feature of animal morphology1-7. Within species, morphological traits need to be correctly proportioned to the body ...

experimental-manipulation-body-size-to-estimate-morphological-scaling

Research

JoVE Journal

Biology

Experimental Manipulation of Body Size to Estimate Morphological Scaling Relationships in Drosophila

13.7K visualizações.  University of Houston. Relações de escala morfológica captar e descrever a forma do organismo. Nós apresentamos um método para medir as relações de escala morfológica em toda a gama natural de tamanhos de corpo dos insetos totalmente metamórfica. Usando uma simples manipulação da dieta que aumentar a distribuição de tamanhos de traço, permitindo a descrição exata de como a forma e tamanho co-variar.

Vídeo: Experimental Manipulation of Body Size to Estimate Morphological Scaling Relationships in Drosophila

Experimental Approaches to Tissue Engineering

Abordagens experimentais de Engenharia de Tecidos

Biologia

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

Imagem Alterar Forma Celular em Viver Drosophila Os embriões

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems including plants, invertebrates and vertebrates. There are various methods to achieve RNAi in vivo4,5. For example, the target gene may be transformed into an RNAi vector, and then either permanently or transiently transformed into cell lines or primary cells to achieve gene knockdown effects; alternatively synthesized double-strand oligonucleotides from specific target genes (RNAi oligos) may be transiently transformed into cell lines or primary cells to silence target genes; or synthesized double-strand RNA molecules may be microinjected into an organism. Since the nematode C. elegans uses bacteria as a food source, feeding the animals with bacteria expressing double-strand RNA against target genes provides a viable strategy6. Here we present an RNAi feeding method to score body size phenotype. Body size in C. elegans is regulated primarily by the TGF- &beta; - like ligand DBL-1, so this assay is appropriate for identification of TGF-&beta; signaling components7. We used different strains including two RNAi hypersensitive strains to repeat the RNAi feeding experiments. Our results showed that rrf-3 strain gave us the best expected RNAi phenotype. The method is easy to perform, reproducible, and easily quantified. Furthermore, our protocol minimizes the use of specialized equipment, so it is suitable for smaller laboratories or those at predominantly undergraduate institutions.

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

We demonstrate how to use the RNAi feeding technique to knock down target genes and score body size phenotype in C. elegans. This method could be used for a large scale screen to identify potential genetic components of interest, such as those involved in body size regulation by DBL-1/TGF-&beta; signaling.

Usando RNA mediada estratégia de alimentação Interferência em tela para genes envolvidos na regulação do tamanho corporal na Nemátodo<em&gt; C. elegans</em

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

Imagens ao vivo de Apuramento celular por apoptose durante<em&gt; Drosophila</em&gt; Embriogênese

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

An Innovative Method for Exosome Quantification and Size Measurement

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about their complex pathways, their bioavailability and their diverse functions in health and disease. Current work focuses on the presence and the behavior of exosomes (in vitro as well as in vivo) in the context of different human disorders, especially in the fields of oncology, gynecology and cardiology.
Unfortunately, neither a consensus regarding a gold standard for exosome isolation exists, nor is there an agreement on such a method for their quantitative analysis. As there are many methods for the purification of exosomes and also many possibilities for their quantitative and qualitative analysis, it is difficult to determine a combination of methods for the ideal approach. 
Here, we demonstrate nanoparticle tracking analysis (NTA), a semi-automated method for the characterization of exosomes after isolation from human plasma by ultracentrifugation. The presented results show that this approach for isolation, as well as the determination of the average number and size of exosomes, delivers reproducible and valid data, as confirmed by other methods, such as scanning electron microscopy (SEM).

A method for isolation of exosomes from whole blood and further analysis by nanoparticle tracking using a semi-automatic instrument is presented in this article. The presented technology provides an extremely sensitive method for visualizing and analyzing particles in liquid suspension.

Um método inovador para exossomo Quantificação e Size Measurement

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about ...

Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory

Emergent properties and external factors (population-level and ecosystem-level interactions, in particular) play important roles in mediating ecologically-important endpoints, though they are rarely considered in toxicological studies. D. melanogaster is emerging as a toxicology model for the behavioral, neurological, and genetic impacts of toxicants, to name a few. More importantly, species in the genus Drosophila can be utilized as a model system for an integrative framework approach to incorporate emergent properties and answer ecologically-relevant questions in&#160;toxicology research. The aim of this paper is to provide a protocol for exposing species in the genus Drosophila to pollutants to be used as a model system for a range of phenotypic outputs and ecologically-relevant questions. More specifically, this protocol can be used to 1) link multiple biological levels of organization and understand the impact of toxicants on both individual- and population-level fitness; 2) test the impact of toxicants at different stages of developmental exposure; 3) test multigenerational and evolutionary implications of pollutants; and 4) test multiple contaminants and stressors simultaneously.

Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory

In this paper, we provide a detailed protocol for exposing species in the genus Drosophila to pollutants with the goal of studying the impact of exposure on a range of phenotypic outputs at different developmental stages and for more than one generation.

Protocolo experimental para o uso de drosófila como um sistema modelo invertebrados para testes de toxicidade em laboratório

Emergent properties and external factors (population-level and ecosystem-level interactions, in particular) play important roles in mediating ...

Noninvasive Monitoring of Lesion Size in a Heterologous Mouse Model of Endometriosis

Here, we describe a protocol for the implementation of a heterologous mouse model in which progression of endometriosis can be assessed in real time through noninvasive monitoring of fluorescence emitted by implanted ectopic human endometrial tissue. For this purpose, biopsies of human endometrium are obtained from donor women ongoing oocyte donation. Human endometrial fragments are cultured in the presence of adenoviruses engineered to express cDNA for the reporter fluorescent protein mCherry. Upon visualization, labeled tissues with an optimal rate of fluorescence after infection are subsequently chosen for the implantation in recipient mice. One week prior to the implantation surgery, recipient mice are oophorectomized, and estradiol pellets are placed subcutaneously to sustain the survival and growth of lesions. On the day of surgery mice are anesthetized, and peritoneal cavity accessed through a small (1.5 cm) incision by the linea-alba. Fluorescently labeled implants are tweezed, briefly soaked in glue and attached to the peritoneal layer. Incisions are sutured, and animals left to recover for a couple of days. Fluorescence emitted by endometriotic implants is usually non-invasively monitored every 3 days for 4 weeks with an in vivo imaging system. Variations in the size of endometriotic implants can be estimated in real time by quantification of the mCherry signal and normalization against the initial time-point showing maximal fluorescence intensity.
Traditional preclinical rodents of models of endometriosis do not allow non-invasive monitoring of lesion in real time but rather allow evaluation of the effects of drugs assayed at the end point. This protocol allows one to track lesions in real time and is more useful to explore the therapeutic potential of drugs in preclinical models of endometriosis. The main limitation of the model thus generated is that non-invasive monitoring is not possible over long periods of time due to the episomal expression of Ad-virus.

Here, we present a protocol for live-imaging of fluorescently labeled human endometrial fragments grafted in mice. The method allows studying the effects of drugs of choice on endometriotic lesion size through monitoring and quantification of fluorescence emitted by the fluorescent reporter on real time

Monitorização não invasiva do tamanho da lesão em um modelo de Mouse heteróloga de endometriose

Here, we describe a protocol for the implementation of a heterologous mouse model in which progression of endometriosis can be assessed in real time ...

Establishment and Genetic Manipulation of Murine Hepatocyte Organoids

The liver is the largest organ in mammals. It plays an important role in glucose storage, protein secretion, metabolism and detoxification. As the executor for most of the liver functions, primary hepatocytes have limited proliferating capacity. This requires the establishment of ex vivo hepatocyte expansion models for liver physiological and pathological research. Here, we isolated murine hepatocytes by two steps of collagenase perfusion and established a 3D organoid culture as the 'mini-liver' to recapitulate cell-cell interactions and physical functions. The organoids consist of heterogeneous cell populations including progenitors and mature hepatocytes. We introduce the process in detailed to isolate and culture the murine hepatocytes or fetal hepatocyte to form organoids within 2-3 weeks and show how to passage them by mechanically pipetting up and down. In addition, we will also introduce how to digest the organoids into single cells for lentivirus infection of shRNA/ectopic construction, siRNA transfection and CRISPR-Cas9 engineering. The organoids can be used for drug screens, disease modelling, and basic liver research by modeling liver biology and pathobiology.

A long-term ex vitro 3D organoid culture system was established from mouse hepatocytes. These organoids can be passaged and genetically manipulated by lentivirus infection of shRNA/ectopic construction, siRNA transfection and CRISPR-Cas9 engineering.

Estabelecimento e Manipulação Genética de Organoides Hepatocitos De Murine

The liver is the largest organ in mammals. It plays an important role in glucose storage, protein secretion, metabolism and detoxification. As the ...

Dissecting, Fixing, and Visualizing the Drosophila Pupal Notum

The pupae of Drosophila melanogaster are immobile for several days during metamorphosis, during which they develop a new body with a thin transparent adult integument. Their immobility and transparency make them ideal for in vivo live imaging experiments. Many studies have focused on the dorsal epithelial monolayer of the pupal&#160;notum because of its accessibility and relatively large size. In addition to the studies of epithelial mechanics and development, the notum has been an ideal tissue to study wound healing. After an injury, the entire epithelial repair process can be captured by live imaging over 6-12 h. Despite the popularity of the notum for live imaging, very few published studies have utilized fixed notum samples. Fixation and staining are common approaches for nearly all other Drosophila tissues, taking advantage of the large repertoire of simple cellular stains and antibodies. However, the pupal notum is fragile and prone to curling and distortion after removal from the body, making it challenging to complement live imaging. This protocol offers a straightforward method for fixing and staining the pupal notum, both intact and after laser-wounding. With this technique, the ventral side of the pupa is glued down to a coverslip to immobilize the pupa, and the notum is carefully removed, fixed, and stained. The notum epithelium is mounted on a slide or between two coverslips to facilitate imaging from the tissue&#39;s dorsal or ventral side.

Dissecting, Fixing, and Visualizing the Drosophila Pupal Notum

The present protocol details the preparation and visualization of fixed tissue of the Drosophila pupal notum. It can be used for either intact or wounded tissue, and the original architecture of the tissue is preserved. The procedures for dissecting, fixing, and staining are all described in this article.

Dissecando, consertando e visualizando o Drosophila Pupal Notum

The pupae of Drosophila melanogaster are immobile for several days during metamorphosis, during which they develop a new body with a thin transparent ...

Induction and Diverse Assessment Indicators of Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is a typical autoimmune disease of the central nervous system (CNS) characterized by inflammatory infiltration, demyelination, and axonal damage. Currently, there are no measures to cure MS completely, but multiple disease-modifying therapies (DMT) are available to control and mitigate disease progression. There are significant similarities between the CNS pathological features of experimental autoimmune encephalomyelitis (EAE) and MS patients. EAE has been widely used as a representative model to determine MS drugs' efficacy and explore the development of new therapies for MS disease. Active induction of EAE in mice has a stable and reproducible effect and is particularly suitable for studying the effects of drugs or genes on autoimmune neuroinflammation. The method of immunizing C57BL/6J mice with myelin oligodendrocyte glycoprotein (MOG35-55) and the daily assessment of disease symptoms using a clinical scoring system is mainly shared. Given the complex etiology of MS with diverse clinical manifestations, the existing clinical scoring system can't satisfy the assessment of disease treatment. To avoid the shortcomings of a single intervention, new indicators to assess EAE based on clinical manifestations of anxiety-like moods and osteoporosis in MS patients are created to provide a more comprehensive assessment of MS treatment.

The present protocol describes the induction of experimental autoimmune encephalomyelitis in a mouse model using myelin oligodendrocyte glycoprotein and monitoring the disease process using a clinical scoring system. Experimental autoimmune encephalomyelitis-related symptoms are analyzed using mouse femur micro-computed tomography analysis and open field test to assess the disease process comprehensively.

Indução e Diversos Indicadores de Avaliação da Encefalomielite Autoimune Experimental

Multiple sclerosis (MS) is a typical autoimmune disease of the central nervous system (CNS) characterized by inflammatory infiltration, demyelination, and ...