Agradecemos a los siguientes organismos de financiación y fundaciones de apoyo: National Institutes of Health, National Science Foundation, la American Heart Association, Asociación de Distrofia Muscular, de la B. Donald y Delia E. Baxter Foundation, el Fondo Klingenstein y la Fundación McKnight para la Neurociencia.

Pipetas tirando El uso de un extractor de microelectrodos como el Sutter P-97 Flaming / Brown, tirar de un conjunto de aproximadamente 10 a 20 pipetas. <ol><li> Seleccione su vaso capilar. Usamos borosilicato capilares (Sutter BF150-110-10, 1,5 mm de diámetro exterior, diámetro interior 1,1 mm, 10 cm de largo). Almacenar el vidrio con cuidado para que permanezca limpia y libre de polvo. </li><li> Diseñar un programa de extracción. Nosotros usamos un programa de cinco pasos, con el calor y la velocidad desciende a cada paso, y un sofá pequeño en el paso final. Libro de cocina de Sutter pipeta es una excelente referencia para el desarrollo de programas adecuados. </li><li> Examine la punta de la pipeta con un microscopio para determinar el diámetro de apertura y suavidad. Deseche las puntas ásperas, desiguales o irregulares. Un buen protocolo tirando debe garantizar que estos son poco frecuentes. Para el estándar de grabación de patch-clamp, las aberturas de punta debe ser 1.3 micrones de diámetro. Un cono empinado (punta roma) conduce a una menor resistencia para el diámetro de apertura. Forma y tamaño pueden ser modificados por la presión de pulido. </li></ol> Pipetas de fuego pulido <ol><li> Establecer una plataforma de pulir (microforge) con un filamento de platino de calefacción controlado por un pedal. Un kit de útiles está disponible a partir de ALA Científicas (CPM-2) o un aparato similar se puede montar poco a poco. </li><li> Opcional: Escudo de la pipeta con un aislante para reducir la capacidad y mejorar las características de ruido. Nosotros usamos la cera dental. Sylgard 184 ™ (poli-dimetil elastómero de siloxano también conocido como PDMS) es también una opción. </li><li> Si se utiliza la cera, mantener un suministro de líquido pequeños cerca. Con la presión del aire en la parte posterior de la pipeta para mantener la cera de entrar en la pipeta, moje la punta brevemente en la cera líquida y remover. Con Sylgard, elastómero tienda preparado en alícuotas congeladas y pintar la punta de la pipeta con un microscopio. Calor para curar el elastómero. </li><li> Coloque la pipeta (recubiertos o no) en el aparato de pulir y llevar la punta ~ 50 micrones del filamento. Tenga en cuenta que el filamento se expande cuando se calienta. </li><li> Recomendado: Siga el protocolo para &quot;pulir la presión&quot; para cambiar la forma de la pipeta para una óptima resistencia y diámetro de la punta. </li><li> Un pulso de calor breve (1-2 segundos) es suficiente para quitar la cera de la punta de la pipeta y el vidrio suave. </li><li> Coloque la pipeta terminó en una caja cerrada para proteger del polvo, de repetición para 10 pipetas de éxito. </li></ol>

El protocolo se ilustra aquí es de uso diario en los laboratorios de electrofisiología, y también se utiliza para fabricar agujas de inyección de células y animales. Con un extractor de programación, es fácil de hacer pipetas para una variedad de usos. Con atención y cuidado, el filamento en su extractor tendrá una duración de un año o más. Buena suerte con sus experimentos.

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<td>Micropipette Puller</td>
<td>Instrument</td>
<td>Sutter Instruments</td>
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<td>Or similar instrument (e.g. Sutter P-87 or P-2000)</td>
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<td>Glass Capillaries</td>
<td>Reagent</td>
<td>Sutter Instruments</td>
<td>BF150-86-10</td>
<td>Or, similar capillary glass. To make filling the pipette easier, use a capillary with a glass filament.</td>
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making patch-pipettes and sharp electrodes with a programmable puller

Microelectrodos de vidrio (también llamado pipetas) han sido un caballo de batalla de la electrofisiología durante décadas. Hoy en día, las pipetas son hechas de capilares de vidrio con un extractor programables. El calor de esos instrumentos el capilar utilizando un filamento de metal o un láser y sacar el cristal con la gravedad, un motor o ambos. Pipetas para la grabación de patch-clamp se forman con el calor y sólo la gravedad, mientras que los electrodos de registro intracelular fuerte uso de una combinación de calor, la gravedad, y un motor. El procedimiento utilizado para pipetas intracelular de grabación es similar a la utilizada para fabricar agujas de inyección para una variedad de aplicaciones, incluyendo la inyección de cRNA en ovocitos de Xenopus. En vidrio en general, capilar &lt;1,2 mm de diámetro se utiliza para hacer las pipetas para la grabación de patch clamp, mientras más estrecho de vidrio se utiliza para el registro intracelular (diámetro externo = 1,0 mm). Para cada herramienta, el tirador está programado de forma ligeramente diferente. Este video muestra cómo hacer que los dos tipos de pipetas de grabación mediante programas preestablecidos extractor.

Watch this Scientific Journal Video about Making Patch-pipettes and Sharp Electrodes with a Programmable Puller at JoVE.com

Making Patch-pipettes and Sharp Electrodes with a Programmable Puller

Este vídeo muestra cómo usar un extractor de programación para pipetas de parches y electrodos para electrofisiología fuerte. El mismo procedimiento puede ser utilizado para hacer una variedad de herramientas de vidrio, incluyendo agujas de inyección.

Hacer patch pipetas y electrodos de Sharp con un extractor programable

Glass microelectrodes (also called pipettes) have been a workhorse of electrophysiology for decades.  Today, such pipettes are made from glass capillaries ...

making-patch-pipettes-and-sharp-electrodes-with-a-programmable-puller

Research

JoVE Journal

Biology

25.3K vistas.  Stanford University. Este vídeo muestra cómo usar un extractor de programación para pipetas de parches y electrodos para electrofisiología fuerte. El mismo procedimiento puede ser utilizado para hacer una variedad de herramientas de vidrio, incluyendo agujas de inyección.

Video: Making Patch-pipettes and Sharp Electrodes with a Programmable Puller

Application of Light-cured Dental Adhesive Resin for Mounting Electrodes or Microdialysis Probes in Chronic Experiments

In chronic recording experiments, self-curing dental acrylic resins have been used as a mounting base of electrodes or microdialysis-probes. Since these acrylics do not bond to the bone, screws have been used as anchors. However, in small experimental animals like finches or mouse, their craniums are very fragile and can not successfully hold the anchors. In this report, we propose a new application of light-curing dental resins for mounting base of electrodes or microdialysis probes in chronic experiments. This material allows direct bonding to the cranium. Therefore, anchor screws are not required and surgical field can be reduced considerably. Past experiences show that the bonding effect maintains more than 2 months. Conventional resin's window of time when the materials are pliable and workable is a few minutes. However, the window of working time for these dental adhesives is significantly wider and adjustable.

In this report, we propose a new application of light-curing dental resins for mounting base of electrodes or microdialysis probes in chronic experiments. This material allows direct bonding to the cranium.

Aplicación de curado con luz Resina Adhesivo Dental para los electrodos de montaje o sondas de microdiálisis en experimentos crónica

In chronic recording experiments, self-curing dental acrylic resins have been used as a mounting base of electrodes or microdialysis-probes. Since these ...

Investigación

Biología

Patterning Cells on Optically Transparent Indium Tin Oxide Electrodes

The ability to exercise precise spatial and temporal control over cell-surface interactions is an important prerequisite to the assembly of multi-cellular constructs serving as in vitro mimics of native tissues.  In this study, photolithography and wet etching techniques were used to fabricate individually addressable indium tin oxide (ITO) electrodes on glass substrates.  The glass substrates containing ITO microelectrodes were modified with poly(ethylene glycol) (PEG) silane to make them protein and cell resistive.  Presence of insulating PEG molecules on the electrode surface was verified by cyclic voltammetry employing potassium ferricyanide as a redox reporter molecule.  Importantly, the application of reductive potential caused desorption of the PEG layer, resulting in regeneration of the conductive electrode surface and appearance of typical ferricyanide redox peaks.    Application of reductive potential also corresponded to switching of ITO electrode properties from cell non-adhesive to cell-adhesive. Electrochemical stripping of PEG-silane layer from ITO microelectrodes allowed for cell adhesion to take place in a spatially defined fashion, with cellular patterns corresponding closely to electrode patterns.  Micropatterning of several cell types was demonstrated on these substrates.   In the future, the control of the biointerfacial properties afforded by this method will allow to engineer cellular microenvironments through the assembly of three or more cell types into a precise geometric configuration on an optically transparent substrate.

Non-fouling PEG silane monolayer was desorbed from individually addressable ITO electrodes on glass by application of a reductive potential. Electrochemical stripping of PEG-silane layer from ITO microelectrodes allowed for cell adhesion to take place in a spatially defined fashion, with cellular patterns corresponding closely to electrode patterns.

Las células de patrón indio electrodos ópticamente transparente de óxido de estaño

The ability to exercise precise spatial and temporal control over cell-surface interactions is an important prerequisite to the assembly of multi-cellular ...

Fabrication of Amperometric Electrodes

Carbon fiber electrodes are crucial for the detection of catecholamine release from vesicles in single cells for amperometry measurements.  Here, we describe the techniques needed to generate low noise (&lt;0.5 pA) electrodes.  The techniques have been modified from published descriptions by previous researchers (1,2).  Electrodes are made by preparing carbon fibers and threading them individually into each capillary tube by using a vacuum with a filter to aspirate the fiber.  Next, the capillary tube with fiber is pulled by an electrode puller, creating two halves, each with a fine-pointed tip.  The electrodes are dipped in hot, liquid epoxy mixed with hardener to create an epoxy-glass seal.  Lastly, the electrodes are placed in an oven to cure the epoxy.  Careful handling of the electrodes is critical to ensure that they are made consistently and without damage.  This protocol shows how to fabricate and cut amperometric electrodes for recording from single cells.

This protocol describes how to generate carbon fiber electrodes. The electrodes are subsequently used to detect catecholamine release from vesicles with carbon fiber amperometry.

Fabricación de electrodos amperométricos

Carbon fiber electrodes are crucial for the detection of catecholamine release from vesicles in single cells for amperometry measurements.  Here, we ...

Brain Banking: Making the Most of your Research Specimens

Unbiased stereology is a method for accurately and efficiently estimating the total neuron number (or other cell type) in a given area of interest1. To achieve this goal 6-10 systematic sections should be probed covering the entire structure. Typically this involves processing 1/5 sections which leaves a significant amount of material unprocessed. In order to maximize the material, we propose an inexpensive method for preserving fixed tissue as part of a long-term storage research plan. As tissue is sliced and processed for the desired stain or antibody, alternate sections should be systematically placed in antigen preserve at -20&deg;C for future processing. Using 24-well plates, sections can be placed in order for future retrieval. Using this method, tissue can be stored and processed for immunohistochemistry over the course of years.

Brain banking and systematic sampling of biological material provides the basis for unbiased stereology and maximizes the potential data obtained from each specimen.

Banca cerebro: Aprovechar al máximo las muestras de su investigación

Unbiased stereology is a method for accurately and efficiently estimating the total neuron number (or other cell type) in a given area of interest1.  To ...

Making MR Imaging Child's Play - Pediatric Neuroimaging Protocol, Guidelines and Procedure

Within the last decade there has been an increase in the use of structural and functional magnetic resonance imaging (fMRI) to investigate the neural basis of human perception, cognition and behavior 1, 2. Moreover, this non-invasive imaging method has grown into a tool for clinicians and researchers to explore typical and atypical brain development. Although advances in neuroimaging tools and techniques are apparent, (f)MRI in young pediatric populations remains relatively infrequent 2. Practical as well as technical challenges when imaging children present clinicians and research teams with a unique set of problems 3, 2. To name just a few, the child participants are challenged by a need for motivation, alertness and cooperation. Anxiety may be an additional factor to be addressed. Researchers or clinicians need to consider time constraints, movement restriction, scanner background noise and unfamiliarity with the MR scanner environment2,4-10. A progressive use of functional and structural neuroimaging in younger age groups, however, could further add to our understanding of brain development. As an example, several research groups are currently working towards early detection of developmental disorders, potentially even before children present associated behavioral characteristics e.g.11. Various strategies and techniques have been reported as a means to ensure comfort and cooperation of young children during neuroimaging sessions. Play therapy 12, behavioral approaches 13, 14,15, 16-18 and simulation 19, the use of mock scanner areas 20,21, basic relaxation 22 and a combination of these techniques 23 have all been shown to improve the participant's compliance and thus MRI data quality. Even more importantly, these strategies have proven to increase the comfort of families and children involved 12. One of the main advances of such techniques for the clinical practice is the possibility of avoiding sedation or general anesthesia (GA) as a way to manage children's compliance during MR imaging sessions 19,20. In the current video report, we present a pediatric neuroimaging protocol with guidelines and procedures that have proven to be successful to date in young children.

Making MR Imaging Child&#039;s Play - Pediatric Neuroimaging Protocol, Guidelines and Procedure

Despite an increase in the use of structural and functional magnetic resonance imaging (fMRI) in humans, the study of young pediatric populations remains a challenge. We present a hands-on, step-by-step video protocol including guidelines for clinicians and researchers intending to perform (f)MRI in young children.

Hacer jugar MR Imaging Niño - Protocolo neuroimagen pediátrica, Directrices y Procedimientos

Within the last decade there has been an increase in the use of structural and functional magnetic resonance imaging (fMRI) to investigate the neural ...

Making Gynogenetic Diploid Zebrafish by Early Pressure

Heterozygosity in diploid eukaryotes often makes genetic studies cumbersome. Methods that produce viable homozygous diploid offspring directly from heterozygous females allow F1 mutagenized females to be screened directly for deleterious mutations in an accelerated forward genetic screen. Streisinger et al.1,2 described methods for making gynogenetic (homozygous) diploid zebrafish by activating zebrafish eggs with ultraviolet light-inactivated sperm and preventing either the second meiotic or the first zygotic cell division using physical treatments (heat or pressure) that deploymerize microtubules. The "early pressure" (EP) method blocks the meiosis II, which occurs shortly after fertilization. The EP method produces a high percentage of viable embryos that can develop to fertile adults of either sex. The method generates embryos that are homozygous at all loci except those that were separated from their centromere by recombination during meiosis I. Homozygous mutations are detected in EP clutches at between 50% for centromeric loci and less than 1% for telomeric loci. This method is reproduced verbatim from the Zebrafish Book3.

This is a method for generating gynogenetic diploid zebrafish embryos (embryos whose only genetic contribution comes from the mother) by blocking the second meiotic division immediately after fertilization with ultraviolet light-inactivated sperm. EP embryos are not fully homozygous due to recombination during the first meiotic division, however they are homozygous at all loci that have not been separated from their centromere by recombination.

Hacer pez cebra Gynogenetic diploides por presión inicial

Heterozygosity in diploid eukaryotes often makes genetic studies cumbersome. Methods that produce viable homozygous diploid offspring directly from ...

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera.


This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time.

Visualización de una sola molécula de ADN de replicación con microscopía de fluorescencia

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated precursors. Researchers have employed various methods of lineage labeling, such as DiI labeling1 and pressure injection of traceable enzymes2 to ascertain cell fate at later stages of development in model systems. The first fate maps in zebrafish (Danio rerio) were assembled by iontophoretic injection of fluorescent dyes, such as rhodamine dextran, into single cells in discrete regions of the embryo and tracing the labeled cell's fate over time3-5. While effective, these methods are technically demanding and require specialized equipment not commonly found in zebrafish labs. Recently, photoconvertable fluorescent proteins, such as Eos and Kaede, which irreversibly switch from green to red fluorescence when exposed to ultraviolet light, are seeing increased use in zebrafish6-8. The optical clarity of the zebrafish embryo and the relative ease of transgenesis have made these particularily attractive tools for lineage labeling and to observe the migration of cells in vivo7. Despite their utility, these proteins have some disadvantages compared to dye-mediated lineage labeling methods. The most crucial is the difficulty we have found in obtaining high 3-D resolution during photoconversion of these proteins. In this light, perhaps the best combination of resolution and ease of use for lineage labeling in zebrafish makes use of caged fluorescein dextran, a fluorescent dye that is bound to a quenching group that masks its fluorescence9. The dye can then be "uncaged" (released from the quenching group) within a specific cell using UV light from a laser or mercury lamp, allowing visualization of its fluorescence or immunodetection. Unlike iontophoretic methods, caged fluorescein can be injected with standard injection apparatuses and uncaged with an epifluorescence microscope equipped with a pinhole10. In addition, antibodies against fluorescein detect only the uncaged form, and the epitope survives fixation well11. Finally, caged fluorescein can be activated with very high 3-D resolution, especially if two-photon microscopy is employed 12,13. This protocol describes a method of lineage labeling by caged fluorescein and laser uncaging. Subsequenctly, uncaged fluorescein is detected simultaneously with other epitopes such as GFP by labeling with antibodies.

This protocol delineates a way to label and trace the fate of small groups of cells zebrafish embryos using UV-uncaging of caged fluorescein, followed by whole mount immunolabeling to amplify the signal from the uncaged fluorescein.

Etiquetado linaje de células de pez cebra con láser Uncagable fluoresceína Dextran

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated ...

Measurement of Cellular Chemotaxis with ECIS/Taxis

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to infection. A common method to measure chemotaxis is the Boyden chamber assay, in which cells and chemoattractant are separated by a porous membrane. As cells migrate through the membrane toward the chemoattractant, they adhere to the underside of the membrane, or fall into the underlying media, and are subsequently stained and visually counted 1. In this method, cells are exposed to a steep and transient chemoattractant gradient, which is thought to be a poor representation of gradients found in tissues 2.
Another assay system, the under-agarose chemotaxis assay, 3, 4 measures cell movement across a solid substrate in a thin aqueous film that forms under the agarose layer. The gradient that develops in the agarose is shallow and is thought to be an appropriate representation of naturally occurring gradients. Chemotaxis can be evaluated by microscopic imaging of the distance traveled. Both the Boyden chamber assay and the under-agarose assay are usually configured as endpoint assays.
The automated ECIS/Taxis system combines the under-agarose approach with Electric Cell-substrate Impedance Sensing (ECIS) 5, 6. In this assay, target electrodes are located in each of 8 chambers. A large counter-electrode runs through each of the 8 chambers (Figure 2). Each chamber is filled with agarose and two small wells are the cut in the agarose on either side of the target electrode. One well is filled with the test cell population, while the other holds the sources of diffusing chemoattractant (Figure 3). Current passed through the system can be used to determine the change in resistance that occurs as cells pass over the target electrode. Cells on the target electrode increase the resistance of the system 6. In addition, rapid fluctuations in the resistance represent changes in the interactions of cells with the electrode surface and are indicative of ongoing cellular shape changes. The ECIS/Taxis system can measure movement of the cell population in real-time over extended periods of time, but is also sensitive enough to detect the arrival of a single cell at the target electrode. 
Dictyostelium discoidium is known to migrate in the presence of a folate gradient 7, 8 and its chemotactic response can be accurately measured by ECIS/Taxis 9. Leukocyte chemotaxis, in response to SDF1&alpha; and to chemotaxis antagonists has also been measured with ECIS/Taxis 10, 11. An example of the leukocyte response to SDF1&alpha; is shown in Figure 1.

The ECIS/Taxis system is an automated, real-time assay that measures cellular chemotaxis. In this assay, cells move beneath a layer of agarose to arrive at a target electrode. Cellular movement is measured by the onset of resistance to AC current 0.

La medición de la quimiotaxis celular con ECIS / taxis

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to ...

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

Many organisms localize mRNAs to specific subcellular destinations to spatially and temporally control gene expression. Recent studies have demonstrated that the majority of the transcriptome is localized to a nonrandom position in cells and embryos. One approach to identify localized mRNAs is to biochemically purify a cellular structure of interest and to identify all associated transcripts. Using recently developed high-throughput sequencing technologies it is now straightforward to identify all RNAs associated with a subcellular structure. To facilitate transcript identification it is necessary to work with an organism with a fully sequenced genome. One attractive system for the biochemical purification of subcellular structures are egg extracts produced from the frog Xenopus laevis. However, X. laevis currently does not have a fully sequenced genome, which hampers transcript identification. In this article we describe a method to produce egg extracts from a related frog, X. tropicalis, that has a fully sequenced genome. We provide details for microtubule polymerization, purification and transcript isolation. While this article describes a specific method for identification of microtubule-associated transcripts, we believe that it will be easily applied to other subcellular structures and will provide a powerful method for identification of localized RNAs.

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

We describe the collection of unfertilized Xenopus tropicalis eggs and production of a meiosis II-arrested egg extract. This egg extract can be used to purify microtubules and microtubule-associated RNAs.

Producción de<em&gt; Xenopus tropicalis</em&gt; Huevos Extrae para identificar los ARN asociada a los microtúbulos

Many  organisms localize mRNAs to specific subcellular destinations to spatially and  temporally control gene expression. Recent studies have demonstrated ...