Name: using a laminating technique to perform confocal microscopy of the human sclera
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German Research Foundation (FOR2240 "(Lymph) Angiogenesis and Cellular Immunity in Inflammatory Diseases of the Eye" to CC and LMH; HE 6743/2-1 and HE 7643/3-1 to LMH; CU47/6-1 to CC), German Cancer Aid (to LMH and CC), GEROK program University of Cologne (to SLS and LMH), and EU COST BM1302 "Joining Forces to Corneal Regeneration" (to CC).

La utilización de tejidos humanos debe ser revisado y aprobado por un comité de revisión institucional o su equivalente. El trabajo aquí descrito fue aprobado por el comité de ética local y tenía la aprobación de examen científico. Este trabajo fue realizado de acuerdo con la Declaración de Helsinki. Los especímenes esclerales humanos se obtuvieron de los ojos de los donantes de globo (máximo post-mortem de tiempo de 24 horas) en el Banco de Ojos del Departamento de Oftalmología de la Universidad de Colonia, Alemania. 1. Preparación Experimental <ol><li> Preparar 96% de etanol y solución salina tamponada con fosfato (PBS) en diferentes tubos. Preparar los anticuerpos primarios en la dilución recomendada de PBS que contiene 2% de albúmina de suero bovino (BSA) y tener todas las soluciones en hielo. Preparar 100 l por muestra. </li><li> Limpiar todos los instrumentos y usar escalpelos afilados. Después de repetir el corte con el mismo bisturí, cambiar el bisturí. </li><li> Obtener 1-2 fórceps Colibri, de rectas tijeras de disección micro, un bisturí # 10o un bisturí oftálmica micro pluma, y ​​cuatro agujas de 26 G. </li><li> Envuelva el papel de aluminio alrededor de una placa de poliestireno o usar una placa de corcho para fijar el tejido. Trabajar debajo de un microscopio estereoscópico binocular. Trabajar estéril debajo de un flujo de aire laminar si es necesario. </li></ol> 2. Preparar la esclerótica <ol><li> coloque un lado del bulbo y realizar una trepanación perforación en la parte corneoscleral, a continuación, girar el trépano. Rotar el trépano con cuidado y de manera uniforme sobre la superficie para realizar un corte por igual todo el año. Use un trépano de tamaño 15,5 mm. Cortar los archivos adjuntos restantes con tijeras curvas. Retire la trepanación corneoscleral. Esto dará lugar a una bombilla en sentido anterior abierto. <ol><li> Ponga la esclerótica en un hisopo con la parte abierta hacia arriba. Retire la retina y la úvea, el uso de fórceps colibri de lograr ambas capas (retina y Uvea) de la esclerótica. No deje la úvea pigmentada sobre la esclerótica interior. Utilice tijeras curvas para eliminar la retina y la úvea de la papila. Retire el remconjuntiva aining, músculos extraoculares, y la cápsula de Tenon de la esclerótica superficial. </li></ol></li><li> Retire la muestra escleral en el tamaño requerido por el uso de las tijeras de tipo recto y las pinzas Colibri. Tomar alrededor de 2 cm 2 muestras de tamaño esclerales de diferentes lugares. Debido a su pequeño tamaño, mantenga la esclerótica con suavidad y cortar al tamaño deseado como cuadrados usando las tijeras de tipo lineal. La trepanación corneoscleral define el margen anterior de las muestras esclerales. <ol><li> Evitar repetida agarrar con el fórceps como el área en la que el tejido fue agarrado no es adecuado para los análisis de microscopía confocal. Preparar el número requerido de muestras en función del tipo experimento (por ejemplo, en sentido anterior vs. posterior, comparar la figura 1) y la cantidad de anticuerpos usados. </li></ol></li><li> Poner las muestras en tubos de 1,5 ml para su uso posterior. Fijar las muestras en 1,5 ml 96% de etanol durante 15 min, y luego los lavar tres veces cada uno durante 5 minutosen 1,5 ml de PBS en el agitador. Si se trabaja con tejido congelado, lleve a cabo este paso antes de la congelación a presión. </li><li> Utilice tejido fresco. Sin embargo, si esto no es posible, broche congelar la esclerótica en nitrógeno líquido y mantenerlo a -20 ° C para su uso posterior. Si se trabaja con tejido congelado, descongelar antes de su uso. Evitar la descongelación repetida y ciclos de congelación. </li><li> Mantener cada muestra en PBS que contenía 1,5 ml 5% de BSA a temperatura ambiente durante 2 hr. Este paso induce una inflamación de la muestra que es útil para la laminación y también evita la unión no específica. </li><li> Después de que las muestras se han hinchado, preparar las plazas esclerales para la laminación. Trabajar por debajo de un microscopio, por ejemplo, un microscopio estéreo binocular. Colocar las muestras esclerales posteriores a la membrana de poliestireno envuelto en papel de aluminio o la placa de corcho utilizando las agujas de 26 G. </li><li> Doblar los bordes de las agujas para que no se interfieren con el campo visual debajo del microscopio. </li><li> Laminar el samp escleral de espesor totalles. <ol><li> En primer lugar, mantenga el borde anterior de la esclerótica utilizando pinzas Colibri. A continuación, cortar cuidadosamente una capa delgada de la esclerótica superficial utilizando el bisturí # 10, la pluma micro bisturí oftálmica, o una hoja de la espátula. Mantenga el bisturí horizontal y cortar la capa superficial lo más finas posible de la capa subyacente. </li><li> Diseccionar una capa delgada escleral de la plaza escleral. Este método es comparable a las técnicas quirúrgicas estándar para preparar un colgajo durante la trabeculectomía y deberían dar lugar a capas gruesas 30-80 micras (comparar Figura 2). </li></ol></li><li> Evitar el secado de la esclerótica mediante la adición de pequeñas cantidades de PBS a los tejidos, por ejemplo, 50 l de PBS utilizando una pipeta. </li><li> Poner las capas cortadas en 100 l de PBS en la placa de 96 pocillos y la etiqueta tanto en la orientación (hacia extern y pasante) y la capa (superficial y profunda), por ejemplo con un color resistente al agua. </li><li> Repita los pasos 2.7-2.9 hasta que el tejido se lamina completamente. &lt;/ Li&gt; </ol> 3. Realizar inmunohistoquímica <ol><li> Añadir 100 l de los anticuerpos primarios requeridos en la dilución recomendada. Aquí, por ejemplo, uso de CD 31 anticuerpo LYVE1 (de conejo anti humano) (ratón monoclonal anti humano) y, tanto en una dilución de 1: 100 en PBS (que contiene 2% de BSA) y se incuba a 4 ° C durante la noche. Para cambiar el medio en la placa de 96 pocillos, mantenga la pipeta a la pared del pozo y extraer el líquido. </li><li> El día siguiente, lavar las muestras tres veces cada una durante 5 minutos con 200 a 300 l de PBS en el agitador. Añadir 100 l de los anticuerpos secundarios correspondientes; aquí utilizar de cabra anti ratón isotiocianato de fluoresceína (FITC) y de cabra anti conejo colorantes de cianina 3 (Cy3), y se incuban las muestras a temperatura ambiente durante 1-2 horas. Diluir los anticuerpos secundarios 1: 300 en PBS que contenía 2% de suero de cabra. </li><li> Enjuagar las muestras de nuevo tres veces cada una durante 5 minutos en 200 a 300 l de PBS en el agitador. </li><li> Realizar tinción núcleo, por ejemplo, </em&gt;. 4 &#39;, 6-diamidino-2-fenilindol (DAPI) (dilución 1: 2000, 100 l en cada pocillo e incubar 10 min a temperatura ambiente), a continuación, lavar de nuevo 2 veces en 200 a 300 l de PBS en el agitador. </li><li> La transferencia de las muestras en portaobjetos de microscopio, incrustarlos en 1-2 gotas de medio de montaje fluorescente, añadir un cubreobjetos, sellarlo, cubriendo los bordes con barniz transparente y se almacena a 4 ° C para su uso posterior o examinar directamente las diapositivas con el confocal microscopio. </li><li> Use un microscopio confocal (o equivalente) para analizar las muestras. Utilice la ampliación deseada, por ejemplo 10-40x ampliación. </li><li> Para verificar el espesor de las muestras, medir con microscopía confocal utilizando Z-pilas. La cantidad de posibles secciones esclerales depende de la ubicación de la esclerótica, como el espesor difiere ecuatorial y posterior (comparar introducción). </li></ol>

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Laminar la esclerótica humana es un método para realizar la microscopía confocal en este tejido. Un paso crítico en este proceso es el uso de etanol en lugar de formalina para fijar el tejido. En nuestra experiencia, los mejores resultados se obtienen cuando se utiliza etanol en lugar de formol para su fijación. escalpelos Blunt agravan el procedimiento y se deben evitar. Del mismo modo, el agotamiento de la esclerótica debe evitarse, ya que complica el procedimiento y reduce la calidad de las imágenes. <p cl...

La esclerótica es la capa exterior rígida que cubre el ojo, que está hecha de tejido conectivo denso. Ayuda a proteger las estructuras intraoculares y para mantener la presión intraocular. Por lo tanto, la esclerótica es esencial para la visión clara. Está desprovisto de vasos linfáticos 1,2 y de ese modo forma un borde libre-linfático exterior entre él y el ojo interior libre de linfático 3-7. También proporciona sitios de unión para los músculos extraoculares, compartiendo así similitudes anatómicas con tendones. Debido a que la esclerótica se compone principalmente de densos haces de colágeno de tipo I y tiene un número menor de tipos de colágeno III, IV, V, VI, VIII 8,9 y elastina 10,11, este tejido no es fácil de usar para inmunohistoquímica. Anatómicamente, la esclerótica se puede separar en tres capas principales: (1) la epiesclerótica vascularizado superficial, que se encuentra debajo de la conjuntiva y la cápsula de Tenon y hacia los lados y THCuenta E del ojo hacia la órbita; (2) el estroma escleral, la parte principal de la esclerótica; y (3) la lámina fusca, que es una capa delgada, pigmentada situado directamente encima de la úvea. Nuestro conocimiento anatómico sobre la esclerótica se deriva principalmente de la primera mitad del siglo 20. En ese momento, los investigadores estudiaron la anatomía de la vasculatura principalmente mediante el uso de inyecciones de tinta India 12 y vasculares fundición 13-15. Más tarde, se investigó en estudios angiográficos 16-19. Desde ese momento, las técnicas más antiguas se han mejorado y otros nuevos han sido desarrollados que nos han permitido complementar los conocimientos anatómicos anterior. Por ejemplo, ha sido solamente alrededor de una década desde que hemos tenido este tipo de marcadores linfáticos fiables como el endotelio vascular linfática hialuronano específico del receptor-1 (LYVE1) 20 o 21 podoplanin. La microscopía confocal ofrece nuevas posibilidades para el estudio de las características anatómicas de los diferentes tiUNDIALES del ojo. Permite múltiples manchas a ser utilizados para diferenciar los marcadores de células o para la localización de las células en relación a los vasos sanguíneos y otras estructuras anatómicas. Proporciona una visión general cuando la muestra es de un tamaño más grande y nos permite escanear a través de una muestra cuando en busca de un tipo específico de célula. Con la tecnología Z-Stack, microscopía confocal se puede utilizar para muestras de hasta 100 a 200 micras. La esclerótica se diferencia de espesor entre 0,3 mm detrás de las inserciones musculares y 1 mm en el polo posterior 11. Debido tanto a su espesor y opacidad, la esclerótica no es adecuado para microscopía confocal utilizando métodos tradicionales. Para remediar esto, los tejidos esclerales se laminaron para permitir su análisis con el microscopio confocal. Esta tecnología es útil para obtener una mejor comprensión de las dos situaciones fisiológicas y patológicas en la esclerótica humana.

<table><tbody><tr><td>96% ethanol</td><td>Merck Chemicals, Darmstadt, Germany</td><td>P075.4</td><td></td></tr><tr><td>binocular stereo microscope&amp;nbsp;</td><td>Motic, Hongkong, China</td><td>n.a</td><td></td></tr><tr><td>26 G needles&amp;nbsp;</td><td>Terumo, Leuven, Belgium</td><td>303800</td><td></td></tr><tr><td>15.5 mm trepan</td><td>Geuder, Heidelberg, Germany</td><td>n.a</td><td></td></tr><tr><td>no.10 scalpel&amp;nbsp;</td><td>Feather, pfm medical, Osaka, Japan</td><td>2E+08</td><td></td></tr><tr><td>ophthalmic scalpel micro feather&amp;nbsp;</td><td>Feather, pfm medical, Osaka, Japan</td><td>no. 7657BR</td><td></td></tr><tr><td>CD 31 antibody (monoclonal mouse anti human)</td><td>Dako, USA</td><td>IR610</td><td></td></tr><tr><td>LYVE1 antibody&amp;nbsp; (polyclonal rabbit anti human)</td><td>Zytomed, Germany</td><td>RBK014-05</td><td></td></tr><tr><td>goat anti mouse FITC antibody</td><td>Sigma Aldrich, Steinheim, Germany</td><td>F0257</td><td></td></tr><tr><td>goat anti rabbit Cy3 antibody</td><td>Dianova, Germany</td><td>111-165-003</td><td></td></tr><tr><td>Goat Serum normal</td><td>Dako, Glostrup, Denmark</td><td>X090710-8</td><td></td></tr><tr><td>DAPI</td><td>Carl Roth GmbH, Karlsruhe, Germany</td><td>6335.1</td><td></td></tr><tr><td>microscope slides&amp;nbsp;</td><td>Engelbrecht, Ederm&amp;uuml;nde, Germany</td><td>WC7695002</td><td></td></tr><tr><td>Coverslips 24 mm x 24 mm</td><td>Th. Gayer, Lohmar, Germany</td><td>7695026</td><td></td></tr><tr><td>DAKO fluorescent mounting medium&amp;nbsp;</td><td>DAKO, USA</td><td>S3023</td><td></td></tr><tr><td>LSM Meta 510 confocal microscopy&amp;nbsp;</td><td>Carl Zeiss AG, Jena, Germany</td><td>n.a</td><td></td></tr></tbody></table>

using a laminating technique to perform confocal microscopy of the human sclera

La esclerótica es un tejido conectivo denso que cubre y protege el ojo. Se compone principalmente de fibras de colágeno denso (tipos I, III, IV, V, VI, y VII). Debido a su autofluorescencia, opacidad, y el espesor, no se ha encontrado adecuado para microscopía confocal. Un enfoque alternativo a la presentada aquí, que utiliza esclerótica fijado con formalina se incluyeron en parafina para inmunohistoquímica, tiene desafíos técnicos, especialmente cuando el precalentamiento del tejido para la recuperación de antígeno. Desde la esclerótica es relativamente pobre en ambas células y vasos, se exploró el uso de muestras de tejido más grandes para ayudar a prevenir que las células vistas y para entender su localización en relación con los buques y otros sitios anatómicos. Para permitir el análisis de muestras de tejido más grandes en el microscopio confocal, una técnica de laminación se realizó para crear capas delgadas de la esclerótica. Tras el análisis de los resultados de los vasos sanguíneos CD31 y hyalu endotelial de vasos linfáticosRonan receptor 1 (LYVE1) células positivas, para lo cual se obtuvo la aprobación para el examen científico, se discuten las ventajas y limitaciones de este método.

En los experimentos representativos realizados aquí, hay beneficios demostrables derivados de la utilización de esta técnica de laminación particular. El primer experimento ilustra la red diversa del plexo vaso sanguíneo episcleral en tres imágenes representativas (figura 3). Los vasos son positivas para CD31. El segundo experimento muestra las células inmunes, en particular las células LYVE1 + de la e...

Watch this Scientific Journal Video about Using a Laminating Technique to Perform Confocal Microscopy of the Human Sclera at JoVE.com

Using a Laminating Technique to Perform Confocal Microscopy of the Human Sclera

Human sclera tissue is mainly collagen; therefore, it is not easily usable for immunohistochemistry. To achieve the goal of performing immunohistochemistry for confocal microscopy of scleral tissue, a laminating technique was used.

Usando una técnica de laminación para realizar la microscopía confocal de la esclerótica humana


The sclera is a dense connective tissue that covers and protects the eye. It mainly consists of dense collagen bundles (types I, III, IV, V, VI, and ...

using-laminating-technique-to-perform-confocal-microscopy-human

Research

JoVE Journal

Medicine

7.5K vistas.  University of Cologne. Human sclera tissue is mainly collagen; therefore, it is not easily usable for immunohistochemistry. To achieve the goal of performing immunohistochemistry for confocal microscopy of scleral tissue, a laminating technique was used. 

Video: Using a Laminating Technique to Perform Confocal Microscopy of the Human Sclera

Three-dimensional Rendering and Analysis of Immunolabeled, Clarified Human Placental Villous Vascular Networks

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that makes up much of the parenchyma of the human placenta. The distal villous capillary network is the terminus of the fetal blood supply after several generations of branching of vessels extending out from the umbilical cord. This network has a contiguous cellular sheath, the syncytial trophoblast barrier layer, which prevents mixing of fetal blood and the maternal blood in which it is continuously bathed. Insults to the integrity of the placental capillary network, occurring in disorders such as maternal diabetes, hypertension and obesity, have consequences that present serious health risks for the fetus, infant, and adult. To better define the structural effects of these insults, a protocol was developed for this study that captures capillary network structure on the order of 1 - 2 mm3 wherein one can investigate its topological features in its full complexity. To accomplish this, clusters of terminal villi from placenta are dissected, and the trophoblast layer and the capillary endothelia are immunolabeled. These samples are then clarified with a new tissue clearing process which makes it possible to acquire confocal image stacks to z- depths of ~1 mm. The three-dimensional renderings of these stacks are then processed and analyzed to generate basic capillary network measures such as volume, number of capillary branches, and capillary branch end points, as validation of the suitability of this approach for capillary network characterization.

This study presents a protocol for the reversible tissue clearing, immunostaining, 3D-rendering and analysis of vascular networks in human placenta villi samples on the order of 1 - 2 mm3.

Representación tridimensional y análisis de Immunolabeled, aclaró redes vasculares vellosas placentarias humanas

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that ...

Investigación

Biología del Desarrollo

Image-guided, Laser-based Fabrication of Vascular-derived Microfluidic Networks

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic networks embedded in PEGDA hydrogels. Here, we describe the creation of virtual masks that allow for image-guided laser control; the photopolymerization of a micromolded PEGDA hydrogel, suitable for microfluidic network fabrication and pressure head-driven flow; the setup and use of a commercially available laser scanning confocal microscope paired with a femtosecond pulsed Ti:S laser to induce hydrogel degradation; and the imaging of fabricated microfluidic networks using fluorescent species and confocal microscopy. Much of the protocol is focused on the proper setup and implementation of the microscope software and microscope macro, as these are crucial steps in using a commercial microscope for microfluidic fabrication purposes that contain a number of intricacies. The image-guided component of this technique allows for the implementation of 3D image stacks or user-generated 3D models, thereby allowing for creative microfluidic design and for the fabrication of complex microfluidic systems of virtually any configuration. With an expected impact in tissue engineering, the methods outlined in this protocol could aid in the fabrication of advanced biomimetic microtissue constructs for organ- and human-on-a-chip devices. By mimicking the complex architecture, tortuosity, size, and density of in vivo vasculature, essential biological transport processes can be replicated in these constructs, leading to more accurate in vitro modeling of drug pharmacokinetics and disease.

This protocol outlines the implementation of image-guided, laser-based hydrogel degradation to fabricate vascular-derived, biomimetic microfluidic networks embedded in poly(ethylene glycol) diacrylate (PEGDA) hydrogels. These biomimetic microfluidic systems may be useful for tissue engineering applications, generation of in vitro disease models, and fabrication of advanced "on-a-chip" devices.

Guiada por imagen, fabricación basada en láser de Redes de microfluidos derivados-vascular

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic ...

Bioingeniería

Light-sheet Microscopy for Three-dimensional Visualization of Human Immune Cells

In vivo, activation, proliferation, and function of immune cells all occur in a three-dimensional (3D) environment, for instance in lymph nodes or tissues. Up to date, most in vitro systems rely on two-dimensional (2D) surfaces, such as cell-culture plates or coverslips. To optimally mimic physiological conditions in vitro, we utilize a simple 3D collagen matrix. Collagen is one of the major components of extracellular matrix (ECM) and has been widely used to constitute 3D matrices. For 3D imaging, the recently developed light-sheet microscopy technology (also referred to as single plane illumination microscopy) is featured with high acquisition speed, large penetration depth, low bleaching, and photocytotoxicity. Furthermore, light-sheet microscopy is particularly advantageous for long-term measurement. Here we describe an optimized protocol how to set up and handle human immune cells, e.g. primary human cytotoxic T lymphocytes (CTL) and natural killer (NK) cells in the 3D collagen matrix for usage with the light-sheet microscopy for live cell imaging and fixed samples. The procedure for image acquisition and analysis of cell migration are presented. A particular focus is given to highlight critical steps and factors for sample preparation and data analysis. This protocol can be employed for other types of suspension cells in a 3D collagen matrix and is not limited to immune cells.

Here, we present a protocol to visualize immune cells embedded in a three-dimensional (3D) collagen matrix using light-sheet microscopy. This protocol also elaborates how to track cell migration in 3D. This protocol can be employed for other types of suspension cells in the 3D matrix.

Microscopía de luz de hoja para la visualización tridimensional de las células inmunes humanas

In vivo, activation, proliferation, and function of immune cells all occur in a three-dimensional (3D) environment, for instance in lymph nodes or ...

Inmunología e Infección

Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking (TXL) for Myopia

Methods to strengthen tissue by introducing chemical bonds (non-enzymatic cross-linking) into structural proteins (fibrillar collagens) for therapy include photochemical cross-linking and tissue cross-linking (TXL) methods. Such methods for inducing mechanical tissue property changes are being employed to the cornea in corneal thinning (mechanically weakened) disorders such as keratoconus as well as the sclera in progressive myopia, where thinning and weakening of the posterior sclera occurs and likely contributes to axial elongation. The primary target proteins for such tissue strengthening are fibrillar collagens which constitute the great majority of dry weight proteins in the cornea and sclera. Fortuitously, fibrillar collagens are the main source of second harmonic generation signals in the tissue extracellular space. Therefore, modifications of the collagen proteins, such as those induced through cross-linking therapies, could potentially be detected and quantitated through the use of second harmonic generation microscopy (SHGM). Monitoring SHGM signals through the use of a laser scanning microscopy system coupled with an infrared excitation light source is an exciting modern imaging method that is enjoying widespread usage in the biomedical sciences. Thus, the present study was undertaken in order to evaluate the use of SHGM microscopy as a means to measure induced cross-linking effects in ex vivo rabbit sclera, following an injection of a chemical cross-linking agent into the sub-Tenon&#39;s space (sT), an injection approach that is standard practice for causing ocular anesthesia during ophthalmologic clinical procedures. The chemical cross-linking agent, sodium hydroxymethylglycinate (SMG), is from a class of cosmetic preservatives known as formaldehyde releasing agents (FARs). Scleral changes following reaction with SMG resulted in increases in SHG signals and correlated with shifts in thermal denaturation temperature, a standard method for evaluating induced tissue cross-linking effects.

Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking TXL for Myopia

This protocol describes techniques for evaluating chemical cross-linking of the rabbit sclera using&#160;second harmonic generation imaging and differential scanning calorimetry.

Segunda generación armónica las señales en esclera de conejo como una herramienta para la evaluación del tejido terapéutico Cross-linking (TXL) para la miopía

Methods to strengthen tissue by introducing chemical bonds (non-enzymatic cross-linking) into structural proteins (fibrillar collagens) for therapy ...

Medicina

<meta charset="utf8"></head><body>Sistema avanzado de cocultivo 3D de dos capas para células epiteliales y fibroblastos

Establishing a Three-Dimensional Coculture Module of Epithelial Cells Using Nanofibrous Membranes

Technical hurdles in a culture of epithelial cells include dedifferentiation and loss of function. Biomimetic three-dimensional (3D) cell culture methods can enhance cell culture efficiency. This study introduces an advanced two-layered culture system intended to cultivate epithelial cells as tissue-like layers with the culture of fibroblasts within a 3D environment. Polyvinyl alcohol (PVA) and poly(&#949;-caprolactone) (PCL) nanofibrous membranes (NMs) were fabricated via electrospinning and utilized as a physiologically relevant extracellular matrix for the culture of epithelial cells and fibroblasts, respectively. In the upper insert wells, lung epithelial cells were cultivated on the PVA NM, and in the lower chambers, fibroblasts were cultured on the PCL NM. This configuration eliminates direct cell-cell contact and facilitates the examination of paracrine signaling mediated by soluble factors. Confocal microscopy was employed to analyze the distribution, growth pattern, and expression of intracellular proteins, including zona occludens in epithelial cells. Z-stacking techniques enabled detailed 3D reconstructions, providing precise insights into the integrity of tight junctions and spatial organization within the epithelial layer. Scanning electron microscopy (SEM) assessed the morphological characteristics of cell types on the nanofibrous membranes. SEM imaging revealed intricate cell surface structures and interactions with the nanofibers, offering a comprehensive perspective on cellular architecture and cell interaction with nanofibrous structure. The Cell Counting Kit-8 (CCK-8) assay is a simple method for measuring epithelial cell and fibroblast growth rates over time. It provides the proliferative behaviors and potential synergistic effects of coculturing these cells. These findings highlight the effectiveness of a simple insert co-culture system for simultaneous culture of fibroblasts and epithelial cells, which is crucial in various physiological and pharmacological contexts, including epithelial tissue regeneration, tumor microenvironment with endothelial, immune, and other stroma cells, toxicity assay, and drug activity test.

<meta charset="utf8"></head><body>Autor destacado: Avance de los sistemas de cocultivo 3D con membranas nanofibrosas de PVA-PCL

Here, we demonstrate how epithelial cells cultured with fibroblasts in the nanofibrous membrane-based two-layer system can stably adhere to and grow on the membrane.

Establecimiento de un módulo de cocultivo tridimensional de células epiteliales utilizando membranas nanofibrosas

Technical hurdles in a culture of epithelial cells include dedifferentiation and loss of function. Biomimetic three-dimensional (3D) cell culture methods ...

Full-Circle Cauterization of Limbal Vascular Plexus for Surgically Induced Glaucoma in Rodents

Glaucoma, the second leading cause of blindness worldwide, is a heterogeneous group of ocular disorders characterized by structural damage to the optic nerve and retinal ganglion cell (RGC) degeneration, resulting in visual dysfunction by interrupting the transmission of visual information from the eye to the brain. Elevated intraocular pressure is the most important risk factor; thus, several models of ocular hypertension have been developed in rodents by either genetic or experimental approaches to investigate the causes and effects of the disease. Among those, some limitations have been reported such as surgical invasiveness, inadequate functional assessment, requirement of extensive training, and highly variable extension of retinal damage. The present work characterizes a simple, low-cost, and efficient method to induce ocular hypertension in rodents, based on low-temperature, full-circle cauterization of the limbal vascular plexus, a major component of aqueous humor drainage. The new model provides a technically easy, noninvasive, and reproducible subacute ocular hypertension, associated with progressive RGC and optic nerve degeneration, and a unique post-operative clinical recovery rate that allows in vivo functional studies by both electrophysiological and behavioral methods.

The goal of this protocol is to characterize a novel model of glaucomatous neurodegeneration based on 360&#176; thermic cauterization of limbal vascular plexus, inducing subacute ocular hypertension.

Cauterización de círculo completo del plexo vascular limbal para el glaucoma inducido quirúrgicamente en roedores

Glaucoma, the second leading cause of blindness worldwide, is a heterogeneous group of ocular disorders characterized by structural damage to the optic ...

Neurociencias

A Tissue Clearing Method for Neuronal Imaging from Mesoscopic to Microscopic Scales

A detailed protocol is provided here to visualize neuronal structures from mesoscopic to microscopic levels in brain tissues. Neuronal structures ranging from neural circuits to subcellular neuronal structures are visualized in mouse brain slices optically cleared with ScaleSF. This clearing method is a modified version of ScaleS and is a hydrophilic tissue clearing method for tissue slices that achieves potent clearing capability as well as a high-level of preservation of fluorescence signals and structural integrity. A customizable three dimensional (3D)-printed imaging chamber is designed for reliable mounting of cleared brain tissues. Mouse brains injected with an adeno-associated virus vector carrying enhanced green fluorescent protein gene were fixed with 4% paraformaldehyde and cut into slices of 1-mm thickness with a vibrating tissue slicer. The brain slices were cleared by following the clearing protocol, which include sequential incubations in three solutions, namely, ScaleS0 solution, phosphate buffer saline (&#8211;), and ScaleS4 solution, for a total of 10.5&#8211;14.5 h. The cleared brain slices were mounted on the imaging chamber and embedded in 1.5% agarose gel dissolved in ScaleS4D25(0) solution. The 3D image acquisition of the slices was carried out using a confocal laser scanning microscope equipped with a multi-immersion objective lens of a long working distance. Beginning with mesoscopic neuronal imaging, we succeeded in visualizing fine subcellular neuronal structures, such as dendritic spines and axonal boutons, in the optically cleared brain slices. This protocol would facilitate understanding of neuronal structures from circuit to subcellular component scales.

The protocol provides a detailed method of neuronal imaging in brain slice using a tissue clearing method, ScaleSF. The protocol includes brain tissue preparation, tissue clarification, handling of cleared slices and confocal laser scanning microscopy imaging of neuronal structures from mesoscopic to microscopic levels.

Un método de limpieza de tejidos para imágenes neuronales de escalas mesoscópicas a microscópicas

A detailed protocol is provided here to visualize neuronal structures from mesoscopic to microscopic levels in brain tissues. Neuronal structures ranging ...

Real Time In Vivo Tracking of Thymocytes in the Anterior Chamber of the Eye by Laser Scanning Microscopy

The purpose of the method being presented is to show, for the first time, the transplant of newborn thymi into the anterior eye chamber of isogenic adult mice for in vivo longitudinal real-time monitoring of thymocytes&#180; dynamics within a vascularized thymus segment. Following the transplantation, laser scanning microscopy (LSM) through the cornea allows in vivo noninvasive repeated imaging at cellular resolution level. Importantly, the approach adds to previous intravital T-cell maturation imaging models the possibility for continuous progenitor cell recruitment and mature T-cell egress recordings in the same animal. Additional advantages of the system are the transparency of the grafted area, permitting macroscopic rapid monitoring of the implanted tissue, and the accessibility to the implant allowing for localized in addition to systemic treatments. The main limitation being the volume of the tissue that fits in the reduced space of the eye chamber which demands for lobe trimming. Organ integrity is maximized by dissecting thymus lobes in patterns previously shown to be functional for mature T-cell production. The technique is potentially suited to interrogate a milieu of medically relevant questions related to thymus function that include autoimmunity, immunodeficiency and central tolerance; processes which remain mechanistically poorly defined. The fine dissection of mechanisms guiding thymocyte migration, differentiation and selection should lead to novel therapeutic strategies targeting developing T cells.

Real Time In Vivo Tracking of Thymocytes in the Anterior Chamber of the Eye by Laser Scanning Microscopy

The goal of the protocol is to show longitudinal intravital real-time tracking of thymocytes by laser scanning microscopy in thymic implants in the anterior chamber of the mouse eye. The transparency of the cornea and vascularization of the graft allows for continuously recording progenitor cell recruitment and mature T-cell egress.

Seguimiento en Vivo de los timocitos en el compartimiento Anterior del ojo mediante láser, microscopía en tiempo real

The purpose of the method being presented is to show, for the first time, the transplant of newborn thymi into the anterior eye chamber of isogenic adult ...

Usando una técnica de laminación para realizar la microscopía confocal de la esclerótica humana

Resumen

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Representación tridimensional y análisis de Immunolabeled, aclaró redes vasculares vellosas placentarias humanas

Guiada por imagen, fabricación basada en láser de Redes de microfluidos derivados-vascular

Microscopía de luz de hoja para la visualización tridimensional de las células inmunes humanas

Segunda generación armónica las señales en esclera de conejo como una herramienta para la evaluación del tejido terapéutico Cross-linking (TXL) para la miopía

Establecimiento de un módulo de cocultivo tridimensional de células epiteliales utilizando membranas nanofibrosas

Establecimiento de un módulo de cocultivo tridimensional de células epiteliales utilizando membranas nanofibrosas

Establecimiento de un módulo de cocultivo tridimensional de células epiteliales utilizando membranas nanofibrosas

Cauterización de círculo completo del plexo vascular limbal para el glaucoma inducido quirúrgicamente en roedores

Un método de limpieza de tejidos para imágenes neuronales de escalas mesoscópicas a microscópicas

Seguimiento en Vivo de los timocitos en el compartimiento Anterior del ojo mediante láser, microscopía en tiempo real