Este trabalho foi financiado por uma concessão do National Science Foundation.

1. 1 dia Antes de começar: Uma população saudável de moscas devem ser mantidas utilizando protocolos padrão de cultura: remove adultos a partir de garrafas ou frascos após 3-4 dias de ovo a postura e permitir o desenvolvimento de proceder a uma temperatura constante até vagando 3 de larvas iniciam pupariation. A transição de larva / pupa é marcado pela formação da pré-pupa (considerado 0 horas após a formação do pupário APF). Pupas imóvel se distinguem dos mais velhos pupas por sua coloração branca e de larvas que ainda não começaram pupariation por seus oblongo, forma arredondada e protrusão do espiráculos anterior. Você vai precisar de: <ul><li> Um pincel para a recolha de pupas </li><li> A câmara úmida por pupas de cultura: A placa de Petri forrada com papel toalha umedecido e coberto com papel de filtro marcado para os pontos de tempo diferentes do genótipo coleção, ou o sexo das pupas </li><li> Fosfato 1X solução salina tamponada (PBS) para lavar pupas </li></ul> Pupas cultura de coleta, e estadiamento <ol><li> Usando um pincel molhado remover suavemente 0hr APF pupas de garrafas de cultura / frascos e coloque-os na tampa da câmara úmida. </li><li> Usando o pincel e 1X PBS lavar cuidadosamente as pupas para remover detritos do caso pupa </li><li> Se necessário, o tipo pupas por sexo. Os primórdios das gônadas de pupas pode ser usado para classificar os sexos. Os primórdios das gônadas masculinas são um par de discos laterais translúcidas facilmente visto através da cutícula pupal aproximadamente 2 / 3 abaixo do comprimento do corpo 5. Os primórdios das gônadas femininas são menores e não tão facilmente identificados. </li><li> Coloque as pupas na posição devidamente rotulados na câmara úmida e voltar à temperatura constante. Cultura ao ponto momento apropriado de desenvolvimento. </li></ol> 2. Dia 2: Dissecção de fixação, e incubação do anticorpo primário Antes de começar: Você vai precisar de: <ul><li> Estereomicroscópio dissecção </li><li> Pinça cirúrgica </li><li> Bisturi cirúrgico com o número 11 lâminas </li><li> Dois de nove pratos de vidro bem depressão (Corning produto # 7220-85) <ul><li> Preencha poços de um prato com 1X PBS: Este é o prato de lavagem para a limpeza de pupas dissecadas </li><li> Preencha poços de segundo prato com tampão de fixação </li></ul></li><li> Câmara úmida para incubação de anticorpos. Nós usamos caixas de plástico selável sanduíche forrada com papel-toalha umedecido. </li><li> Buffer de fixação: 1x PBS, paraformaldeído 4%, 0,2% de ácido deoxicólico (para permeabilização) </li><li> Plataforma de dissecção: lâmina de microscópio Limpar com um pedaço de fita dupla face adere a um dos lados </li><li> 1X PBS </li><li> p100 ou p200 pipetador </li></ul> Dissecação <ol><li> Utilizando uma pinça remova cuidadosamente uma pupa de cada vez e coloque-os sobre a plataforma de dissecção com a extremidade anterior do pupas de frente para a maior largura da fita. Se pupas são excessivamente molhada, primeiro blot-os secar com uma toalha de papel. </li><li> Antes de as pupas foram completamente aderida à fita, use um pincel para posicioná-los. </li><li> Permitir que as pupas secar ao ar e aderem à fita (5-10 minutos) </li><li> Com o bisturi cirúrgico dissecar cada pupas bilateralmente. Este é o melhor feito com um único corte rápido da anterior para a extremidade posterior da pupa. Se feito corretamente, o corte será bisect pares anterior e posterior espiráculos. Dissecar não mais de 10 pupas de cada vez como lavagem e limpeza das amostras rapidamente é necessário para evitar danos proteolíticas para o tecido. </li><li> Usando um pincel, transferir uma pequena quantidade de 1X PBS a cada pupas dissecadas para soltá-las a partir da fita. </li></ol> Fixação de limpeza, e incubação do anticorpo primário <ol start="6"><li> Com um par de pinças cirúrgicas agarrar um meia pupa individuais anteriormente e imediatamente mergulhe em um poço de lavar o prato. A plataforma de dissecção deve ser substituído por o prato de lavagem no palco microscópio. O caso pupas devem ser deixados sobre a amostra até que o processamento está completo e as amostras estão prontas para montagem. </li><li> Enquanto ainda segurando a meia pupa use uma pipeta suavemente para lavar o tecido interno do abdômen. Muita pressão durante a lavagem pode levar à perda de células epiteliais. </li><li> Transferir imediatamente a metade pupa limpa para o buffer de fixação à temperatura ambiente e processo as metades restantes pupa da mesma forma. Com dissecção prática, e de lavagem de 20 metades pupa deve levar menos de 5 minutos. </li><li> Permitir pupas para incubar em tampão de fixação em temperatura ambiente por 1 (uma) hora. </li><li> Enxágüe fixos pupas 3x cinco minutos em PBS 1X </li><li> As amostras podem ser imediatamente processadas para imunohistoquímica ou podem ser armazenados em etanol a 100% a -20 ° C por até 3 meses sem perda de células ou reatividade epítopo. </li><li> Para armazenar amostras, equilibrar as amostras através de uma diluiçãosérie de PBS: EtOH (3:1, 1:1, 1:3) à temperatura ambiente por 20 minutos em cada diluição antes de transferir até 100% EtOH. </li><li> As amostras devem ser re-equilibrados em PBS através da série de reverter antes do processamento de imuno-histoquímica. </li><li> Bloco de amostras em 1x PBS (suplementado com 2% de albumina bovina sérica) para 1 (uma) hora antes da adição do anticorpo primário. </li><li> Incubar com anticorpo primário diluído a concentração adequada em 1X PBS durante a noite a 4 ° C sem balançar. </li></ol> 3. Dia 3: a incubação do anticorpo secundário, a montagem ea imagem Antes de começar: Você vai precisar de: <ul><li> Duas pinças cirúrgicas </li><li> Meios de montagem (glicerol, Vectashield [Vector Labs], ou Slowfade Ouro [Invitrogen]) </li><li> Slides depressão bem microscópio (0,8 mm) </li><li> Lamelas (24 x 40 mm) </li></ul> Incubação do anticorpo secundário e montagem: <ol><li> Lavar as amostras de 10 minutos cada um com 3x 1x PBS </li><li> Bloco de amostras em 1x PBS (suplementado com 2% de albumina bovina sérica) para 1 (uma) hora antes da adição do anticorpo secundário. </li><li> Incubar com anticorpo secundário diluído a concentração adequada em 1X PBS a temperatura ambiente no escuro sem balançar por 3 (três) horas. </li><li> Lavar as amostras de 10 minutos cada um com 3x 1x PBS </li><li> Se for o caso, para contraste pupas (Exemplo nós mancha núcleos com DAPI [4 &#39;,6-diamidino-2-phenylindole] diluído em PBS durante 10 minutos). </li><li> Equilibrar as amostras (mínimo de 30 minutos) em mídia apropriada antes da montagem. </li><li> Prepare uma lâmina de amostra. A morfologia das pupas impede de montagem plana. Amostras são montados no poço de uma lâmina de depressão (slide drop) para geração de imagens. Lugar de 75 100ul de meios de montagem na depressão também. Várias amostras podem ser montados em um único slide. </li><li> O caso pupa deve ser removido das amostras antes da montagem. Segure um caso pupa individuais anteriormente tendo a certeza de não compreender a membrana interna pupal. </li><li> Com um segundo par de fórceps gentilmente compreender a membrana interna pupal pela cabeça e removê-lo do caso pupal. </li><li> Transferir imediatamente a amostra para a depressão bem e continuar o processamento de amostras adicionais. </li><li> Usando uma posição da sonda ou pinça as amostras uniformemente na depressão bem com a superfície lateral voltada para cima. Bolhas de ar ocasionais podem ser removidos com uma sonda. </li><li> Abaixe suavemente a lamínula sobre as amostras tomando cuidado para não introduzir bolhas de ar </li></ol> 4. Resultados representativos: Amostras preparadas usando este protocolo reter a morfologia bruta do abdômen adulto. Pilhas de imagens podem ser projetadas para gerar uma imagem bidimensional ou 3-D de renderização pode ser aplicado para investigar topologia abdômen. <img src="/files/ftp_upload/3139/3139fig1.jpg" alt="Figura 1"/> Figura 1. Produtos segmentação gene em Drosophila pupas de proteínas e expressão Wingless Engrailed (En-gal4: uas-GFP). Foram visualizados em 26 horas após a formação pupário (APF) com o mouse anti-Wg (4D4: Iowa Hibridoma Bank) e anti-GFP. Ampliação de 10x, é anterior à esquerda e dorsal é para cima. Núcleos com DAPI foram contra. Pilhas de cerca de 50 imagens (ΔZ entre fatias é 2.5μm) foram projetados.

<ol>
	<li>Kopp, A., Duncan, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anteroposterior+patterning+in+adult+abdominal+segments+of+Drosophila.">Anteroposterior patterning in adult abdominal segments of Drosophila.</a> Dev. Biol. 242, 15-30 (2002).</li><li>Ninov, N., Chiarelli, D. A., Martin-Blanco, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extrinsic+and+intrinsic+mechanisms+directing+epithelial+cell+sheet+replacement+during+Drosophila+metamorphosis.">Extrinsic and intrinsic mechanisms directing epithelial cell sheet replacement during Drosophila metamorphosis.</a> Development. 134, 367-379 (2007).</li><li>Bischoff, M., Cseresnyes, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+rearrangements,+cell+divisions+and+cell+death+in+a+migrating+epithelial+sheet+in+the+abdomen+of+Drosophila.">Cell rearrangements, cell divisions and cell death in a migrating epithelial sheet in the abdomen of Drosophila.</a> Development. 136, 2403-2411 (2009).</li><li>Matsuda, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=in+Morphology+and+evolution+of+the+insect+abdomen.">in Morphology and evolution of the insect abdomen.</a> , (1976).</li><li>Kerkis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Growth+of+the+Gonads+in+Drosophila+Melanogaster.">The Growth of the Gonads in Drosophila Melanogaster.</a> Genetics. 16, 212-224 (1931).</li></ol>

Não há conflitos de interesse declarados.

As técnicas apresentadas neste vídeo pode ser usado para preparar Drosophila pupas de uma variedade de pontos de tempo de desenvolvimento. Pupas processadas durante o período de 24 horas a 32 horas APF APF são mais propensas a perda de células do epitélio. O uso de detergentes (como Triton X-100 e Tween-20) durante as etapas de incubação prolongado aumenta a probabilidade de perda de células e não é recomendada. Pelo contrário, ácido deoxicólico é usado como um detergente durante a etapa de fixação inicial. Todas as etapas subseqüentes são realizados em 1X PBS sem detergente. Além disso, balançando amostras durante as etapas de incubação prolongado aumenta a perda de células e devem ser evitados. As amostras podem ser fotografada usando técnicas de microscopia confocal. No entanto, Drosophila pupas processado como descrito também pode ser fotografada usando um sistema de microscopia de iluminação estruturada produzindo qualidade de imagem comparável às técnicas confocal.

<ul><li> Estereomicroscópio dissecção </li><li> Pinça cirúrgica </li><li> Bisturi cirúrgico com o número 11 lâminas </li><li> Nove pratos de vidro bem depressão (Corning produto # 7220-85) </li><li> Câmara úmida por pupas de cultura </li><li> Fosfato 1X solução salina tamponada (PBS) </li><li> # 11 bisturi cirúrgico </li><li> fita dupla face </li><li> Buffer de fixação: 1x PBS, paraformaldeído 4%, 0,2% de ácido deoxicólico (para permeabilização) </li><li> p100 ou p200 pipetador </li><li> Slides depressão bem microscópio (0,8 mm) </li></ul>

drosophila pupal abdomen immunohistochemistry

A Drosophila abdômen pupal é um sistema modelo estabelecido para o estudo da morfogênese epitelial e desenvolvimento de morfologias sexualmente dimórfico 1-3. Durante a pupação, que se estende por aproximadamente 96 horas (a 25 ° C), as populações de proliferação das células da epiderme imaginal substituir larval para gerar os segmentos adulto abdominal. Estas células imaginal, nascido durante a embriogênese, existem como pares laterais de ninhos histoblast em cada segmento abdominal das larvas. Quatro pares de ninhos histoblast dar lugar à cutícula do adulto dorsal (anterior e posterior ninhos dorsal), a cutícula ventral (ninhos ventral) e os espiráculos associados a cada segmento (ninhos espiráculo) 4. Após puparation, essas células diplóides (distinguíveis pelo tamanho da maior poliplóide larval células epidérmicas-LECs) iniciar um processo estereotipada da migração, proliferação e substituição da LECs. Várias ferramentas moleculares e genéticos podem ser empregados para investigar as contribuições dos caminhos genéticos envolvidos na morfogênese do abdômen adulto. Fenótipos adultos final são tipicamente analisados ​​dissecção seguintes adulto cutículas abdominal. No entanto, a investigação dos processos moleculares subjacentes requer análises imuno-histoquímico do epitélio pupal, que apresentam desafios únicos. Temporalmente morfogênese dinâmica e as interações de duas populações distintas epiteliais (larval e imaginal) geram um tecido frágil propensos à perda excessiva de células durante a dissecção e processamento subseqüente. Nós desenvolvemos métodos de dissecação, fixação, montagem e criação de imagens do epitélio pupal abdominem Drosophila para estudos de imuno-histoquímica que geram amostras consistentes de alta qualidade adequado para confocal ou padrão de microscopia fluorescente.

Watch this Scientific Journal Video about Drosophila Pupal Abdomen Immunohistochemistry at JoVE.com

Drosophila Pupal Abdomen Immunohistochemistry

Coloração do anticorpo<em&gt; Drosophila</em&gt; Pupas pode melhorar análises genéticas de adultos abdominal genética do desenvolvimento. Apresentamos nosso protocolo para a fixação de dissecação, e coloração de anticorpos encenado<em&gt; Drosophila</em&gt; Abdome pupal.

Drosophila Imunohistoquímica Abdomen Pupal

The Drosophila pupal abdomen is an established model system for the study of epithelial morphogenesis and the development of sexually dimorphic ...

drosophila-pupal-abdomen-immunohistochemistry

Research

JoVE Journal

Biology

Drosophila Pupal Abdomen Immunohistochemistry

16.1K visualizações.  University of Alabama. Coloração do anticorpo Drosophila Pupas pode melhorar análises genéticas de adultos abdominal genética do desenvolvimento. Apresentamos nosso protocolo para a fixação de dissecação, e coloração de anticorpos encenado Drosophila Abdome pupal.

Vídeo: Drosophila Pupal Abdomen Immunohistochemistry

Thin Sectioning of Slice Preparations for Immunohistochemistry

Many investigations in neuroscience, as well as other disciplines, involve studying small, yet macroscopic pieces or sections of tissue that have been preserved, freshly removed, or excised but kept viable, as in slice preparations of brain tissue.  Subsequent microscopic studies of this material can be challenging, as the tissue samples may be difficult to handle.  Demonstrated here is a method for obtaining thin cryostat sections of tissue with a thickness that may range from 0.2-5.0 mm.  We routinely cut 400 micron thick Vibratome brain slices serially into 5-10 micron coronal cryostat sections.  The slices are typically first used for electrophysiology experiments and then require microscopic analysis of the cytoarchitecture of the region from which the recordings were observed.  We have constructed a simple device that allows controlled and reproducible preparation and positioning of the tissue slice.  This device consists of a cylinder 5 cm in length with a diameter of 1.2 cm, which serves as a freezing stage for the slice.  A ring snugly slides over the cylinder providing walls around the slice allowing the tissue to be immersed in freezing compound (e.g., OCT).  This is then quickly frozen with crushed dry ice and the resulting  wafer  can be position easily for cryostat sectioning.  Thin sections can be thaw-mounted onto coated slides to allow further studies to be performed, such as various staining methods, in situ hybridization, or immunohistochemistry, as demonstrated here.

The present method allows reproducible cryostat sectioning of small, difficult-to-manage, tissue pieces, such as biopsies and brain slices. We utilize a simple aluminum freezing stage to facilitate handling of tissue and a standard cryostat to routinely produce 5-10 micron serial sections from 400 micron thick brain slices.

Seccionamento fina de Preparações Slice para Imunohistoquímica

Many investigations in neuroscience, as well as other disciplines, involve studying small, yet macroscopic pieces or sections of tissue that have been ...

Biologia

In situ Protocol for Butterfly Pupal Wings Using Riboprobes

Here we present, in video format, a protocol for in situ hybridizations in pupal wings of the butterfly Bicyclus anynana using riboprobes.  In situ hybridizations, a mainstay of developmental biology, are useful to study the spatial and temporal patterns of gene expression in developing tissues at the level of transcription. If antibodies that target the protein products of gene transcription have not yet been developed, and/or there are multiple gene copies of a particular protein in the genome that cannot be differentiated using available antibodies, in situs can be used instead. While an in situ technique for larval wing discs has been available to the butterfly community for several years, the current protocol has been optimized for the larger and more fragile pupal wings.

In order to examine gene expression in the pupal wing tissue of Bicyclus anynana, we present an optimized protocol for in situ hybridizations using riboprobes. We also provide guidelines for the further optimization of this protocol for use in pupal wings of other Lepidopteran species.

No Protocolo situ de asas da borboleta Pupal Usando Riboprobes

Here we present, in video format, a protocol for in situ hybridizations in pupal wings of the butterfly Bicyclus anynana using riboprobes.  In situ ...

Immunohistochemistry: Paraffin Sections Using the Vectastain ABC Kit from Vector Labs

Immunohistochemistry (IHC) is a valuable technique utilized to localize/visualize protein expression in a mounted tissue section using specific antibodies. There are two methods: the direct and indirect method. In this experiment, we will only describe the use of indirect IHC staining. Indirect IHC staining utilizes highly specific primary and biotin-conjugated secondary antibodies. Primary antibodies are utilized to discretely identify proteins of interest by binding to a specific epitope, while secondary antibodies subtract for non-specific background staining and amplify signal by forming complexes to the primary antibody. Slides can either be generated from frozen sections, or paraffin embedded sections mounted on glass slides. In this protocol, we discuss the preparation of paraffin-embedded sections by dewaxing, hydration using an alcohol gradient, heat induced antigen retrieval, and blocking of endogenous peroxidase activity and non-specific binding sites. Some sections are then stained with antibodies specific for T cell marker CD8 and while others are stained for tyrosine hydroxylase. The slides are subsequently treated with appropriate secondary antibodies conjugated to biotin, then developed utilizing avidin-conjugated horseradish peroxidase (HRP) with Diaminiobenzidine (DAB) as substrate. Following development, the slides are counterstained for contrast, and mounted under coverslips with permount. After adequate drying, these slides are then ready for imaging.

Imuno-histoquímica: Seções de parafina Usando o Kit de Vectastain ABC Vector Labs

Immunohistochemistry (IHC) is a valuable technique utilized to localize/visualize protein expression in a mounted tissue section using specific ...

Immunohistochemistry on Paraffin Sections of Mouse Epidermis Using Fluorescent Antibodies

In the epidermis, immunohistochemistry is an efficient means of localizing specific proteins to their relative expression compartment; namely the basal, suprabasal, and stratum corneum layers. The precise localization within the epidermis of a particular protein lends clues toward its functional role within the epidermis. In this chapter, we describe a reliable method for immunolocalization within the epidermis modified for both frozen and paraffin sections that we use very routinely in our laboratory. Paraffin sections generally provide much better morphology, hence, superior results and photographs; however, not all antibodies will work with the harsh fixation and treatment involved in their processing. Therefore, the protocol for frozen sectioning is also included. Within paraffin sectioning, two fixation protocols are described (Bouin's and paraformaldehyde); the choice of fixative will be directly related to the antibody specifications and may require another fixing method.

We describe a reliable method for immunolocalization within the epidermis modified for both frozen and paraffin sections that we use very routinely in our laboratory.

Imuno-histoquímica em parafina da epiderme do rato utilizando anticorpos fluorescentes

In the epidermis, immunohistochemistry is an efficient means of localizing specific proteins to their relative expression compartment; namely the basal, ...

Drosophila Larval NMJ Immunohistochemistry

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 31 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. Immunohistochemistry can be used to clearly image the components of the NMJ. In this article, we demonstrate how to use antibody staining to visualize the Drosophila larval NMJ.

Drosophila Larval NMJ Immunohistochemistry

This protocol demonstrates how to perform immunohistochemistry on dissected Drosophila larva.

Drosophila larval MNJ Imunohistoquímica

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use ...

Production of Tissue Microarrays, Immunohistochemistry Staining and Digitalization Within the Human Protein Atlas

The tissue microarray (TMA) technology provides the means for high-throughput analysis of multiple tissues and cells. The technique is used within the Human Protein Atlas project for global analysis of protein expression patterns in normal human tissues, cancer and cell lines. Here we present the assembly of 1 mm cores, retrieved from microscopically selected representative tissues, into a single recipient TMA block. The number and size of cores in a TMA block can be varied from approximately forty 2 mm cores to hundreds of 0.6 mm cores. The advantage of using TMA technology is that large amount of data can rapidly be obtained using a single immunostaining protocol to avoid experimental variability. Importantly, only limited amount of scarce tissue is needed, which allows for the analysis of large patient cohorts 1 2. Approximately 250 consecutive sections (4 &mu;m thick) can be cut from a TMA block and used for immunohistochemical staining to determine specific protein expression patterns for 250 different antibodies. In the Human Protein Atlas project, antibodies are generated towards all human proteins and used to acquire corresponding protein profiles in both normal human tissues from 144 individuals and cancer tissues from 216 different patients, representing the 20 most common forms of human cancer. Immunohistochemically stained TMA sections on glass slides are scanned to create high-resolution images from which pathologists can interpret and annotate the outcome of immunohistochemistry. Images together with corresponding pathology-based annotation data are made publically available for the research community through the Human Protein Atlas portal (<a href="http://www.proteinatlas.org" target="_blank">www.proteinatlas.org</a>) (Figure 1) 3 4. The Human Protein Atlas provides a map showing the distribution and relative abundance of proteins in the human body. The current version contains over 11 million images with protein expression data for 12.238 unique proteins, corresponding to more than 61% of all proteins encoded by the human genome.

Tissue microarrays allows for an efficient method to gain concurrent information from a multitude of tissues. Representative parts of tissues are assembled into a single paraffin block. Sections from the block are used for immunohistochemistry and analysis of protein expression patterns. Digital scanning generates corresponding images for distribution of data.

Produção de Microarrays Tecido, de coloração imuno-histoquímica e de digitalização Dentro do Atlas proteína humana

The  tissue microarray (TMA) technology provides the means for high-throughput  analysis of multiple tissues and cells. The technique is used within the ...

Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ring-supported hydrophilized PTFE membrane to provide structural support to both living and fixed tissue during immunohistochemical processing, which allows for the use of a variety of protocols that would otherwise cause damage to the tissue. First, this is demonstrated with bolus loading of fluorescent markers into living retinal tissue. This method allows for quick visualization of targeted structures, while the membrane support maintains tissue integrity during the injection and allows for easy transfer of the preparation for further imaging or processing.
Second, a procedure for antibody staining in tissue fixed with carbodiimide is described. Though paraformaldehyde fixation is more common, carbodiimide fixation provides a superior substrate for the visualization of synaptic proteins. A limitation of carbodiimide is that the resulting fixed tissue is relatively fragile; however, this is overcome with the use of the supporting membrane. Retinal tissue is used to demonstrate these techniques, but they may be applied to any fragile tissue.

The protocol described here is for structural assessment of a wholemount retinal preparation. This includes descriptions of tissue dissection, mounting onto a hydrophilized polytetrafluoroethylene (PTFE) membrane insert, bolus loading with fluorescent markers, and a comparison of fixation with carbodiimide and paraformaldehyde for immunohistochemical analysis of cellular and synaptic components.

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ...

Imaging Through the Pupal Case of Drosophila melanogaster

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled analysis of cell and developmental biology from a variety of methodological angles. Live imaging is an emerging method for observing dynamic cell processes, such as cell division or cell motility. Having isolated mutations in uncharacterized putative cell cycle proteins it became essential to observe mitosis in situ using live imaging. Most live imaging studies in Drosophila have focused on the embryonic stages that are accessible to manipulation and observation because of their small size and optical clarity. However, in these stages the cell cycle is unusual in that it lacks one or both of the gap phases. By contrast, cells of the pupal wing of Drosophila have a typical cell cycle and undergo a period of rapid mitosis spanning about 20 hr of pupal development. It is easy to identify and isolate pupae of the appropriate stage to catch mitosis in situ. Mounting intact pupae provided the best combination of tractability and durability during imaging, allowing experiments to run for several hours with minimal impact on cell and animal viability. The method allows observation of features as small as, or smaller than, fly chromosomes. Adjustment of microscope settings and the details of mounting, allowed extension of the preparation to visualize membrane dynamics of adjacent cells and fluorescently labeled proteins such as tubulin. This method works for all tested fluorescent proteins and can capture submicron scale features over a variety of time scales. While limited to the outer 20 &#181;m of the pupa with a conventional confocal microscope, this approach to observing protein and cellular dynamics in pupal tissues in vivo may be generally useful in the study of cell and developmental biology in these tissues.

Imaging Through the Pupal Case of Drosophila melanogaster

This paper demonstrates the use of a fast scanning confocal microscope to image cell behavior directly through the puparium. By leaving the pupal case intact, this method allows observation and measurement of dynamic cell processes at a stage of Drosophila development that is difficult to study directly.

Imagem Através do Pupal Caso de<em&gt; Drosophila melanogaster</em

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled ...

Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment

During spermatogenesis in mammals and in Drosophila melanogaster, male germ cells develop in a series of essential developmental processes. This includes differentiation from a stem cell population, mitotic amplification, and meiosis. In addition, post-meiotic germ cells undergo a dramatic morphological reshaping process as well as a global epigenetic reconfiguration of the germ line chromatin&#8212;the histone-to-protamine switch.
Studying the role of a protein in post-meiotic spermatogenesis using mutagenesis or other genetic tools is often impeded by essential embryonic, pre-meiotic, or meiotic functions of the protein under investigation. The post-meiotic phenotype of a mutant of such a protein could be obscured through an earlier developmental block, or the interpretation of the phenotype could be complicated. The model organism Drosophila melanogaster offers a bypass to this problem: intact testes and even cysts of germ cells dissected from early pupae are able to develop ex vivo in culture medium. Making use of such cultures allows microscopic imaging of living germ cells in testes and of germ-line cysts. Importantly, the cultivated testes and germ cells also become accessible to pharmacological inhibitors, thereby permitting manipulation of enzymatic functions during spermatogenesis, including post-meiotic stages.
The protocol presented describes how to dissect and cultivate pupal testes and germ-line cysts. Information on the development of pupal testes and culture conditions are provided alongside microscope imaging data of live testes and germ-line cysts in culture. We also describe a pharmacological assay to study post-meiotic spermatogenesis, exemplified by an assay targeting the histone-to-protamine switch using the histone acetyltransferase inhibitor anacardic acid. In principle, this cultivation method could be adapted to address many other research questions in pre- and post-meiotic spermatogenesis.

Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment

This protocol describes the dissection and cultivation of intact testes and germ-line cysts from Drosophila melanogaster pupae. This method allows microscopic observation of spermatogenesis ex vivo. Furthermore, we describe a pharmacological assay of the effect of inhibitors on specific stages of germ-cell development in pupal testes.

Ex vivo Cultura de Drosophila Pupal Testículo e único macho da linha germinal Cistos: Dissection, Imaging, eo tratamento farmacológico

During spermatogenesis in mammals and in Drosophila melanogaster, male germ cells develop in a series of essential developmental processes. This includes ...

Dissection and Mounting of Drosophila Pupal Eye Discs

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes. It contains approximately 800 separate eye units, termed ommatidia1. Each ommatidium contains eight neuronal photoreceptors that develop from undifferentiated cells following the passage of the morphogenetic furrow in the third larval instar2. Following the sequential differentiation of the photoreceptors, non-neuronal cells develop, including cone and pigment cells, along with mechanosensory bristle cells3. Final differentiation processes, including the structured arrangement of all the ommatidial cell types, programmed cell death of undifferentiated cell types and rhodopsin expression, occurs through the pupal phase4-7. This technique focuses on manipulating the pupal eye disc, providing insight and instruction on how to dissect the eye disc during the pupal phase, which is inherently more difficult to perform than the commonly dissected third instar eye disc. This technique also provides details on immunostaining to allow the visualization of various proteins and other cell components.

Dissection and Mounting of Drosophila Pupal Eye Discs

The goal of this technique is to enable researchers to perform dissection, immunostaining and mounting of pupal eye discs from Drosophila melanogaster of any age.

Dissecção e montagem de<em&gt; Drosophila</em&gt; pupais Discos Eye

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes.  It contains approximately 800 ...