Este trabajo fue apoyado por una beca de la Fundación Nacional de Ciencias.

1. Día 1 Antes de empezar: Una población sana de la mosca se debe mantener a través de protocolos estándar de cultivo: eliminar adultos a partir de botellas o frascos después de 3-4 días de huevo-laicos y permitir el desarrollo de proceder a una temperatura constante hasta que vagar 3 de larvas iniciar pupariation. La transición de larva / pupa se caracteriza por la formación de la prepupas (considera 0 horas después de la formación de pupa-APF). Pupas inmóviles se distinguen de los mayores pupas por su coloración blanca y de las larvas que aún no han comenzado pupariation por su alargada, forma redondeada y saliente de los espiráculos anteriores. Usted necesitará: <ul><li> Un pincel para recoger las pupas </li><li> Una cámara húmeda para pupas cultivo: una placa de Petri llenas de toalla de papel húmeda y cubierta con papel de filtro para el marcado varios momentos de la recogida, el genotipo o el sexo de las pupas </li><li> 1X fosfato (PBS) para el lavado de las pupas </li></ul> Recolección, el cultivo y la puesta en escena pupas <ol><li> Con un pincel mojado retire suavemente 0hr pupas APF de las botellas de la cultura / viales y colóquelos en la tapa de la cámara húmeda. </li><li> Utilizando el pincel y PBS 1X lavar suavemente las pupas para eliminar los restos de la caja pupa </li><li> Si es necesario ordenar las pupas por sexo. Los primordios de las gónadas de las pupas se pueden utilizar para ordenar los sexos. El macho primordios gonadales son un par de discos lateral translúcida fácilmente visible a través de la cutícula pupal aproximadamente 2 / 3 a lo largo del cuerpo 5. La hembra primordios gonadales son más pequeños y no se identifican con la misma facilidad. </li><li> Colocar las pupas en la posición estén debidamente etiquetados en la cámara húmeda y se vuelva a la temperatura constante. La cultura hasta el punto de tiempo de desarrollo apropiado. </li></ol> 2. Día 2: Disección, fijación e incubación del anticuerpo primario Antes de empezar: Usted necesitará: <ul><li> La disección estereoscópica </li><li> Fórceps quirúrgicos </li><li> Bisturí quirúrgico con el número 11 hojas </li><li> Dos de nueve y platos de cristal depresión (Corning # 7220-85) <ul><li> Llenar los pozos de una placa con PBS 1X: Este es el plato lavado para la limpieza de las pupas disecados </li><li> Llenar los pozos de segundo plato con el tampón de fijación </li></ul></li><li> Cámara húmeda para la incubación de anticuerpos. Utilizamos cajas de plástico herméticos sándwich cubierto con toallas de papel húmedas. </li><li> Tampón de fijación: 1x PBS, paraformaldehído al 4%, 0,2% de ácido desoxicólico (para la permeabilización) </li><li> Plataforma de disección: portaobjetos abierto con un trozo de cinta de doble cara adherido a un lado </li><li> 1X PBS </li><li> p100 o p200 pipeta </li></ul> Disección <ol><li> El uso de pinzas retire suavemente una pupa en un momento y lugar en la plataforma de disección con el extremo anterior de la pupa se enfrenta el amplio ancho de la cinta. Si pupas son excesivamente húmedo, primera mancha secar con una toalla de papel. </li><li> Antes de las pupas tienen completamente adherido a la cinta, utilice una brocha para la posición. </li><li> Permita que las pupas se sequen al aire y se adhieren a la cinta (5-10 minutos) </li><li> Con el bisturí quirúrgico diseccionar cada pupas bilateral. Esto se logra mejor con un solo corte rápido de la parte anterior del extremo posterior de la pupa. Si se hace correctamente, el corte será dividir en dos pares de espiráculos anteriores y posteriores. Diseccionar no más de 10 pupas en un momento como el lavado y limpieza de las muestras con rapidez es necesaria para evitar daños a los tejidos proteolíticas. </li><li> Usando un pincel, la transferencia de una pequeña cantidad de PBS 1X a cada pupas disecados para evitar que se peguen a la cinta. </li></ol> Limpieza, fijación y la incubación del anticuerpo primario <ol start="6"><li> Con un par de pinzas quirúrgicas captar un individuo medio pupa anterior e inmediatamente se sumergen en un pozo de la placa de enjuague. La plataforma de la disección debe ser sustituido por el plato de enjuague en la platina del microscopio. El caso de las pupas se debe dejar en la muestra hasta que se complete el proceso y las muestras están listas para el montaje. </li><li> Aunque sin soltar la mitad pupa utilizar una pipeta para lavar suavemente el tejido interno del abdomen. El exceso de presión durante el lavado se puede llevar a la pérdida de las células epiteliales. </li><li> Inmediatamente la transferencia de la limpieza medio pupa para amortiguar la fijación a temperatura ambiente y el proceso de los restantes pupa mitades de manera similar. Con la práctica, la disección y el lavado de 20 mitades pupa debe tener menos de 5 minutos. </li><li> Permita que las pupas de incubación en tampón de fijación a temperatura ambiente durante 1 (una) hora. </li><li> Enjuague fija pupas 3x cinco minutos en PBS 1X </li><li> Las muestras pueden ser inmediatamente procesados ​​para inmunohistoquímica o se puede almacenar en el 100% de etanol a -20 ° C hasta por 3 meses sin pérdida de las células o reactividad epítopo. </li><li> Para almacenar las muestras, las muestras se equilibran a través de una diluciónserie de PBS: EtOH (3:1, 1:1, 1:3) a temperatura ambiente durante 20 minutos en cada dilución antes de transferirlos al EtOH al 100%. </li><li> Las muestras deben ser re-equilibrada en PBS a través de la serie antes de la transformación inversa para inmunohistoquímica. </li><li> Bloque de muestras en 1x PBS (complementado con bovinos 2% de albúmina sérica) a 1 (una) hora antes de la adición del anticuerpo primario. </li><li> Se incuba con el anticuerpo primario diluido a la concentración adecuada en 1X PBS durante la noche a 4 ° C, sin balanceo. </li></ol> 3. Día 3: incubación del anticuerpo secundario, el montaje y la imagen Antes de empezar: Usted necesitará: <ul><li> Dos pinzas quirúrgicas </li><li> Medios de montaje (glicerol, Vectashield [Vector Labs], o Slowfade Oro [Invitrogen]) </li><li> Diapositivas depresión microscopio y (0,8 mm) </li><li> Cubreobjetos (24 x 40 mm) </li></ul> Incubación del anticuerpo secundario y montaje: <ol><li> Lávese las muestras de 3x 10 minutos cada uno con PBS 1x </li><li> Bloque de muestras en 1x PBS (complementado con bovinos 2% de albúmina sérica) a 1 (una) hora antes de la adición del anticuerpo secundario. </li><li> Incubar con el anticuerpo secundario diluido a la concentración adecuada en 1X PBS a temperatura ambiente en la oscuridad sin balanceo de 3 (tres) horas. </li><li> Lávese las muestras de 3x 10 minutos cada uno con PBS 1x </li><li> En su caso, de contraste pupas (Ejemplo que mancha los núcleos con DAPI [4 &#39;,6-diamidino-2-phenylindole] diluido en PBS durante 10 minutos). </li><li> Equilibrar las muestras (un mínimo de 30 minutos) en un medio adecuado antes de montar. </li><li> Prepare una diapositiva de muestra. La morfología de las pupas impide el montaje plano. Las muestras se montan en el pozo de la depresión de una diapositiva (slide drop) para obtener imágenes. Lugar de 75 100 ul de medio de montaje en la depresión también. Varias muestras pueden ser montados en una sola diapositiva. </li><li> El caso de pupa se debe quitar de las muestras antes de su montaje. Tome un individuo pupa caso anterior asegurándose de no comprender la membrana interna de pupa. </li><li> Con un segundo par de pinzas de agarre suave de la membrana interna de las pupas de la cabeza y sacarlo de la envoltura pupal. </li><li> Inmediatamente la transferencia de la muestra a la depresión, así y continuar el procesamiento de muestras adicionales. </li><li> Usando una posición de la sonda o con pinzas las muestras de manera uniforme en la depresión y con la superficie lateral hacia arriba. Burbujas de vez en cuando el aire puede ser removido con una sonda. </li><li> Baje suavemente el cubreobjetos sobre las muestras con cuidado de no introducir burbujas de aire </li></ol> 4. Los resultados representativos: Las muestras preparadas con este protocolo conservar la morfología general del abdomen adulto. Las pilas de la imagen puede ser proyectada para generar una imagen de dos dimensiones o de la prestación en 3-D puede ser aplicado para investigar la topología de abdomen. <img src="/files/ftp_upload/3139/3139fig1.jpg" alt="Figura 1"/> Figura 1. Productos de la segmentación de genes en Drosophila pupas proteína sin alas y la expresión Engrailed (In-GAL4: UAS-GFP). Se visualizaron en 26 horas después de la formación pupario (APF) con el ratón anti-WG (4D4: Iowa hibridoma Banco) y anti-GFP. Un aumento de 10x, es anterior a la izquierda y dorsal está arriba. Núcleos se counterstained con DAPI. Las pilas de aproximadamente 50 imágenes (ΔZ entre rebanadas es 2.5μm) fueron proyectados.

<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>

No hay conflictos de interés declarado.

Las técnicas presentadas en este video se puede utilizar para preparar Drosophila pupa de una variedad de puntos de tiempo de desarrollo. Pupas procesado durante el período de 24 horas a 32 horas APF APF son más propensos a la pérdida de células del epitelio. El uso de detergentes (como el Triton X-100 y Tween-20) durante las etapas de incubación prolongado aumenta la probabilidad de pérdida de células y por lo tanto no se recomienda. Por el contrario, ácido desoxicólico se utiliza como un detergente durante la etapa de fijación inicial. Todos los pasos posteriores se realizan en PBS 1X sin detergente. Además, meciéndose muestras durante las etapas de incubación prolongado aumenta la pérdida de células y se debe evitar. Las muestras pueden tomarse imágenes mediante técnicas de microscopía confocal. Sin embargo, Drosophila pupas procesaron como se describe también se pueden visualizar mediante un sistema de iluminación microscopía estructurado dando una calidad de imagen comparable a las técnicas de confocal.

<ul><li> La disección estereoscópica </li><li> Fórceps quirúrgicos </li><li> Bisturí quirúrgico con el número 11 hojas </li><li> Nueve y platos de cristal depresión (Corning producto # 7220-85) </li><li> Cámara húmeda para el cultivo de pupas </li><li> 1X fosfato (PBS) </li><li> # 11 bisturí quirúrgico </li><li> cinta de doble cara </li><li> Tampón de fijación: 1x PBS, paraformaldehído al 4%, 0,2% de ácido desoxicólico (para la permeabilización) </li><li> p100 o p200 pipeta </li><li> Diapositivas depresión microscopio y (0,8 mm) </li></ul>

drosophila pupal abdomen immunohistochemistry

El abdomen de Drosophila pupa es un sistema modelo establecido para el estudio de la morfogénesis del epitelio y el desarrollo de morfologías sexualmente dimórficos 1-3. Durante la fase de pupa, que se extiende por aproximadamente 96 horas (a 25 ° C), las poblaciones de la proliferación de las células imaginales reemplazar la epidermis larval para generar los segmentos abdominales adulto. Estas células imaginal, nacido durante la embriogénesis, existen como pares de nidos histoblast lateral en cada segmento abdominal de las larvas. Cuatro pares de nidos histoblast dar lugar a la cutícula del adulto dorsal (nidos dorsal anterior y posterior), la cutícula ventral (nidos ventral) y los espiráculos asociados a cada segmento (nidos espiráculo) 4. Al puparation, estas células diploides (que se distingue por el tamaño de las larvas más grandes poliploides células epidérmicas-LEC) iniciar un proceso estereotipado de la proliferación, la migración y la sustitución de la LEC. Varias herramientas moleculares y genéticos pueden ser empleados para investigar la contribución de mecanismos genéticos implicados en la morfogénesis del abdomen adulto. Fenotipos última adultos suelen ser analizado después de la disección de los adultos cutículas abdominal. Sin embargo, la investigación de los procesos moleculares que subyacen requiere un análisis inmunohistoquímico del epitelio de pupa, que presentan desafíos únicos. Temporalmente la morfogénesis dinámica y las interacciones de dos poblaciones distintas epiteliales (las larvas y imaginal) generar un tejido frágiles propensas a la pérdida excesiva de células durante la disección y posterior procesamiento. Hemos desarrollado métodos de disección, fijación, montaje y la imagen del epitelio abdominem Drosophila pupa para estudios inmunohistoquímicos que generan consistentes muestras de alta calidad adecuada para la microscopía de fluorescencia confocal o estándar.

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

Drosophila Pupal Abdomen Immunohistochemistry

Tinción de los anticuerpos<em&gt; Drosophila</em&gt; Pupas pueden mejorar los análisis genéticos de los adultos la genética del desarrollo abdominal. Se presenta el protocolo para la tinción de la disección, la fijación de anticuerpos y de escena<em&gt; Drosophila</em&gt; Pupal abdomen.

Drosophila pupa inmunohistoquímica Abdomen

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 vistas.  University of Alabama. Tinción de los anticuerpos Drosophila Pupas pueden mejorar los análisis genéticos de los adultos la genética del desarrollo abdominal. Se presenta el protocolo para la tinción de la disección, la fijación de anticuerpos y de escena Drosophila Pupal abdomen.

Video: 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.

El corte delgado de los preparativos de círculo para inmunohistoquímica

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

Investigación

Biología

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.

En el Protocolo situ de Alas de Mariposa pupa Con ribosondas

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.

Inmunohistoquímica: las secciones de parafina utilizando la Vectastain ABC Kit de 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.

Inmunohistoquímica en secciones de parafina de la epidermis del ratón Utilizando anticuerpos 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 NMJ inmunohistoquímica

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.

La producción de microarrays de tejidos, las técnicas de inmunohistoquímica y la digitalización En el 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 ...

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.

Protocolo optimizado para Retina Preparación Wholemount de la Imagen y la inmunohistoquímica

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.

Imaging A través de la envoltura pupal 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 Testis y individuales masculinos quistes línea germinal: Disección, Imaging y Tratamiento 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.

Disección y montaje de<em&gt; Drosophila</em&gt; Pupal 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 ...