El trabajo en la investigación del ojo en este laboratorio con el apoyo de la Fundación Panamericana de la Salud Auxiliar, Azalea (1972) Fondo de Dotación y el Fondo de Pequeños Proyectos HKU (20097176185).

1. Instrumentos Microscopio, Nikon Investigador de música, MBF Bioscience - MicroBrightField, Inc. 2. Preparación <ol><li> Antes de trabajar en cualquier análisis morfométricos, cada muestra de retina es seccionadas a 4 micras de espesor y se somete a la tinción H &amp; E. </li><li> La sección de la retina se divide en 4 regiones: región periférica superior, la región central superior, la región periférica inferior, y la región central inferior. </li><li> Después de definir las regiones, una imagen de cada región se adquiere en las ampliaciones 10x40 bajo el microscopio de haz brillante de Nikon. </li><li> Ahora, estamos listos para analizar la morfometría de la retina. </li></ol> 3. Medición de espesor INL <ol><li> Cuatro mediciones del espesor del INL en cada región se midieron. Cuatro líneas se extraerán perpendicularmente desde la línea de base del contorno de la frontera exterior de la INL. </li><li>Abra el software llamado Investigador estéreo y elegir el lente de 40x de la barra de herramientas principal. </li><li> Seleccione &quot;Abrir imagen&quot; en la pestaña &quot;Archivo&quot;. Elija una imagen de una región que desee la retina que le gustaría analizar. </li><li> Elija el tipo de línea de contorno del cuadro de lista desplegable de la barra de herramientas principal y pulse el botón &quot;Mover automático&quot;. </li><li> Haga clic derecho en la ventana de localización, y seleccione &quot;Simple Haga clic en localización&quot;. </li><li> Dibuje la curva de nivel a lo largo del borde interior de la INL. </li><li> Cuando el dibujo se hace, haga clic derecho en la ventana de la búsqueda y elegir la opción &quot;Contorno Open End&quot;. </li><li> Seleccione un marcador en la barra de herramientas de marcadores y añadir marcadores en los puntos de unión. </li><li> Después de añadir todos los marcadores, deseleccionar el icono de marcador en la barra de herramientas de marcadores. </li><li> Coloque un cursor en el centro de uno de los marcadores. </li></ol><img src="/files/ftp_upload/3377/3377fig1.jpg" alt="Figura 1" /><ol start="11"><li> Seleccione &quot;QuickMedir ángulo &quot;en la pestaña&quot; Herramientas &quot;. </li></ol><img src="/files/ftp_upload/3377/3377fig2.jpg" alt="Figura 2" /><ol start="12"><li> Haga clic derecho en la ventana de localización y anular la selección del &quot;continuo&quot; opción. </li><li> Medir un ángulo por primera haciendo clic en el centro de un marcador, que es adyacente al marcador seleccionado para un extremo del segmento de línea. </li></ol><img src="/files/ftp_upload/3377/3377fig3.jpg" alt="Figura 3" /><ol start="14"><li> A continuación, mueva el ratón hacia el centro de la marca en cuestión y haga clic de nuevo. </li><li> Extender la línea de banda de goma de la frontera exterior de la INL. </li><li> Ajuste el ángulo de hasta 90 ° se alcanza. </li></ol><img src="/files/ftp_upload/3377/3377fig4.jpg" alt="Figura 4" /><ol start="17"><li> Clic izquierdo en la posición deseada. Un cuadro de diálogo que muestra una medición de ángulo aparecerá justo después del clic. </li><li> Pulse el botónTecla &quot;Enter&quot; mientras se mantiene el ratón todavía. </li></ol><img src="/files/ftp_upload/3377/3377fig5.jpg" alt="Figura 5" /><ol start="19"><li> Haz clic izquierdo en la ventana de localización de manera que se dibuja una línea desde un extremo hasta el final elegido nuevo. </li><li> Haga clic derecho en la ventana de la búsqueda y elegir la opción &quot;Contorno Open End&quot;. </li></ol><img src="/files/ftp_upload/3377/3377fig6.jpg" alt="Figura 6" /><ol start="21"><li> Dibujar las otras líneas en las posiciones diferentes en la misma forma como se ha descrito antes. </li><li> Después de dibujar todas las líneas, seleccionar el modo de edición y cambiar el nombre de los 4 segmentos de línea en consecuencia. </li><li> Cuatro mediciones se puede obtener pulsando el botón &quot;Medición de contorno&quot; botón. </li><li> Seleccione estas 4 medidas y pegarlos en una hoja de cálculo haciendo clic en &quot;Copiar al portapapeles&quot;. </li><li> Seleccione &quot;Guardar como&quot; en la pestaña &quot;Archivo&quot;. Los datos de seguimiento se pueden guardar como un archivo de datos. </li></ol><ol><li> Seleccione &quot;Abrir imagen&quot; en la pestaña &quot;Archivo&quot;. Elija una imagen de una región que desee la retina que le gustaría analizar. </li><li> Elija el tipo de línea de contorno del cuadro de lista desplegable de la barra de herramientas principal. </li><li> Dibuja una línea de contorno a lo largo de la frontera de la capa de fibras nerviosas (NFL). </li><li> Poner fin a la búsqueda en la &quot;Contorno Open End&quot; opción. </li><li> En cada extremo, añadir un marcador en la punta de la línea y otro en la posición cerca de la punta. </li><li> Desactive el icono de marcador en la barra de herramientas de marcadores. </li><li> Coloque un cursor en el centro de los marcadores en la punta. </li><li> Seleccione &quot;Ángulo Medida rápida&quot; en la pestaña &quot;Herramientas&quot;. </li><li> Haga clic derecho en la ventana de localización y anular la selección del &quot;continuo&quot; opción. </li><li> Ajuste las líneas de banda de goma y medir un ángulo de 90 °. </li><li> Pulse la tecla &quot;Enter&quot; después de un cuadro de diálogo está elevado. </li><li> Izquierda haga clic en tél rastreo ventana de modo que una nueva línea puede ser extendido perpendicularmente desde la punta. </li><li> Dibuje otra línea nueva en la misma manera en el otro extremo. </li><li> Dos límites son creados dentro de la cual el número de CGR se cuenta. </li><li> Elija una frontera como una línea de exclusión que los toques de células no se cuenta. </li><li> Seleccione un tipo de marcador y colocar uno a cada célula viva. </li><li> Seleccione otro tipo de marcador de las células muertas. </li><li> El número de cada tipo de marcadores colocados se mostrará junto al icono de marcador en consecuencia en la barra de herramientas de marcadores. </li><li> Seleccione el modo de edición y recoger la línea de base del contorno. Haga clic derecho en la ventana de localización y cambiar el nombre de la línea. </li><li> La longitud de la línea de base del contorno puede ser obtenido mediante prensado &quot;Medida del contorno&quot; botón. </li><li> El número de las células pueden ser expresados ​​por mm de la longitud. </li><li> Seleccione &quot;Guardar como&quot; en la pestaña &quot;Archivo&quot;. Lala localización y los datos de marcadores se pueden guardar como un archivo de datos. </li></ol> 5. La medición del tamaño de la celda de una celda CGR <ol><li> Seleccione &quot;Abrir imagen&quot; en la pestaña &quot;Archivo&quot;. Elija una imagen de una región que desee la retina que le gustaría analizar. </li><li> Ampliar en una célula viva y elegir el tipo de línea de contorno del cuadro de lista desplegable de la barra de herramientas principal. </li><li> Dibujar una línea de contorno a lo largo de la circunferencia de la célula. </li><li> Casi al final, haga clic derecho en la ventana de localización y seleccionar &quot;End contorno de cerca&quot;. </li><li> El área de la celda se muestra con el tema del área en el cuadro de diálogo de medición Contour. </li></ol>

<ol>
	<li>Lam, T. T., Abler, A. S., Tso, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-Methyl-D-Aspartate+(NMDA)â&#x20AC;&#x201C;Induced+Apoptosis+in+Rat+Retina.">N-Methyl-D-Aspartate (NMDA)â&#x20AC;&#x201C;Induced Apoptosis in Rat Retina.</a> Investigative Ophthalmology &amp; Visual Science. 40, 2391-2397 (1999).</li><li>Lam, T. T., Abler, A. S., Tso, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptosis+and+caspases+after+ischemia-reperfusion+injury+in+rat+retina.">Apoptosis and caspases after ischemia-reperfusion injury in rat retina.</a> Invest. Ophthalmol. Vis. Sci. 40, 967-975 (1999).</li><li>Luo, X. G., Chiu, K., Lau, H. S., Lee, V. W. H., Yung, K. K. L., So, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Selective+Vulnerability+of+Retinal+Ganglion+Cells+in+Rat+Chronic+Ocular+Hypertension+Model+at+Early+Phase.">The Selective Vulnerability of Retinal Ganglion Cells in Rat Chronic Ocular Hypertension Model at Early Phase.</a> Cellular and Molecular Neurobiology. 29 (8), 1143-1151 (2009).</li><li>Han, Y. S., Chung, I. Y., Park, J. M., Yu, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroprotective+effect+of+citicoline+on+retinal+cell+damage+induced+by+kainic+acid+in+rats.">Neuroprotective effect of citicoline on retinal cell damage induced by kainic acid in rats.</a> Korean J. Ophthalmol. 19, 219-226 (2005).</li><li>Hernandez, M., Urcola, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+ganglion+cell+neuroprotection+in+a+rat+model+of+glaucoma+following+brimonidine,+latanoprost+or+combined+treatments.">Retinal ganglion cell neuroprotection in a rat model of glaucoma following brimonidine, latanoprost or combined treatments.</a> Exp. Eye Res. 86, 798-806 (2008).</li><li>Chan, H. C., Chang, R. C. C., Ip, A. K. C., Chiu, K., Yuen, W. H., Zee, S. Y., So, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroprotective+effects+of+Lycium+barbarum+Lynn+on+protecting+retinal+ganglion+cells+in+an+ocular+hypertension+model+of+glaucoma.">Neuroprotective effects of Lycium barbarum Lynn on protecting retinal ganglion cells in an ocular hypertension model of glaucoma.</a> Experimental Neurology. 203, 269-273 (2007).</li></ol>

Los autores no tienen nada que revelar.

1. Cómo obtener una medición de espesores más precisa? Puede ampliar la imagen haciendo clic en &quot;Zoom&quot; y la izquierda haga clic en la ventana de seguimiento. El borde INL puede verse claramente. Si la punta de la línea no está en la frontera exterior de la INL, podemos ajustar la línea en el modo de edición. Después de recoger la línea en cuestión, haga clic derecho en la ventana de la búsqueda y elegir la opción &quot;Punto de inserción en el contorno seleccionado&quot;....

A correction was made to <a href="http://www.jove.com/video/3377">Morphometric Analyses of Retinal Sections</a>. There was an error with an author's name.

Erratum: Morphometric Analyses of Retinal Sections

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Tipo </td><td> Empresa </td><td> Número de catálogo </td></tr><tr><td> Investigador estéreo </td><td> MBF Software de Análisis de Biociencias </td><td> Campo MicroBright </td><td></td></tr><tr><td> Microscopio </td><td> Olympus BX51 </td><td> Olympus Corporation </td><td> BX51 </td></tr></tbody></table>

morphometric analyses of retinal sections

El análisis morfométrico de secciones de retina se han utilizado en el examen de enfermedades de la retina. Por ejemplos, las células neuronales se perdieron significativamente en la capa de células ganglionares de retina (RGCL) en modelos de ratas con N-metil-D-aspartato (NMDA)-inducido excitotoxicidad 1, la retina isquemia-reperfusión 2 y 3 del glaucoma. Reducción de la capa plexiforme INL e interior (IPL) de espesor se revirtieron con el tratamiento con citicolina en los ojos de las ratas sometidas a la excitotoxicidad del glutamato kaínico mediada por el ácido 4. La alteración de la densidad de CGR y los tamaños de soma se observaron con los tratamientos farmacológicos diferentes en los ojos con presión intraocular elevada 3,5,6. Por lo tanto, con métodos objetivos de análisis de los morfometría retinianos pueden ser de gran importancia en la evaluación de patologías de la retina y la eficacia de las estrategias terapéuticas. La estructura de la retina es multi-capas y diferentes tipos de neuronas exiº en la retina. Los parámetros morfométricos de la retina, tales como el número de células, tamaño de la celda y el espesor de las capas diferentes son más complejos que el sistema de cultivo celular. Al principio, estos parámetros pueden ser detectados utilizando otro software de imagen comercial. Los valores son normalmente de valor relativo, y cambiando el valor exacto puede ser necesario el cálculo más exacto. Además, el trazado del tamaño celular y la morfología no puede ser precisa y suficientemente sensible como para el análisis estadístico, especialmente en el modelo de glaucoma crónico. Las medidas utilizadas en este protocolo siempre de una manera más precisa y fácil. Y la longitud absoluta de la línea y el tamaño de la celda se puede informar directamente y fácil de ser copiado en otros archivos. Por ejemplo, se trazó el margen de los núcleos más interior y exterior en el INL y formó una línea a continuación, utilizando el software para dibujar un ángulo de 90 grados para medir el espesor. Aunque sin la ayuda del software, la línea oblicua tal y el cambio de espesor de la retina puedeno ser repetible entre los observadores individuales. Además, el número y la densidad de CGR también puede ser cuantificado. Este protocolo con éxito disminuye la variabilidad en la cuantificación de las características de la retina, aumenta la sensibilidad en la detección de cambios mínimos. Este vídeo se muestran tres tipos de análisis morfométricos de las secciones de la retina. Estas incluyen la medición del espesor de la INL, que cuantifica el número de las células y medir el tamaño de las CGR en valor absoluto. Estos tres análisis se llevan a cabo con el investigador estéreo (MBF Bioscience - MicroBrightField, Inc.). La técnica puede ofrecer una plataforma sencilla pero científica de análisis morfométricos.

Watch this Scientific Journal Video about Morphometric Analyses of Retinal Sections at JoVE.com

Morphometric Analyses of Retinal Sections

Este video muestra tres tipos de análisis morfométricos de la retina, que incluyen la medición del espesor de la capa interna nuclear, cuantificar el número de células ganglionares de la retina (CGR) y la medición de los tamaños de las CGR. La técnica puede ofrecer una plataforma sencilla pero científica de análisis morfométricos.

Los análisis morfométricos de las Secciones de retina

Morphometric analyses of retinal sections have been used in examining retinal diseases. For examples, neuronal cells were significantly lost in the ...

morphometric-analyses-of-retinal-sections

Research

JoVE Journal

Neuroscience

10.2K vistas.  The University of Hong Kong. Este video muestra tres tipos de análisis morfométricos de la retina, que incluyen la medición del espesor de la capa interna nuclear, cuantificar el número de células ganglionares de la retina (CGR) y la medición de los tamaños de las CGR. La técnica puede ofrecer una plataforma sencilla pero científica de análisis morfométricos.

Video: Morphometric Analyses of Retinal Sections

An Isolated Retinal Preparation to Record Light Response from Genetically Labeled Retinal Ganglion Cells

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1. This information is then segregated into what are known as the ON and OFF pathways. The photoreceptors signal light information to the bipolar cells (BCs), which depolarize in response to increases (On BCs) or decreases (Off BCs) in light intensity. This segregation of light information is maintained at the level of the retinal ganglion cells (RGCs), which have dendrites stratifying in either the Off sublamina of the inner plexiform layer (IPL), where they receive direct excitatory input from Off BCs, or stratifying in the On sublamina of the IPL, where they receive direct excitatory input from On BCs. This segregation of information regarding increases or decreases in illumination (the On and Off pathways) is conserved and signaled to the brain in parallel.
The RGCs are the output cells of the retina, and are thus an important cell to study in order to understand how light information is signaled to visual nuclei in the brain. Advances in mouse genetics over recent decades have resulted in a variety of fluorescent reporter mouse lines where specific RGC populations are labeled with a fluorescent protein to allow for identification of RGC subtypes 2 3 4 and specific targeting for electrophysiological recording. Here, we present a method for recording light responses from fluorescently labeled ganglion cells in an intact, isolated retinal preparation. This isolated retinal preparation allows for recordings from RGCs where the dendritic arbor is intact and the inputs across the entire RGC dendritic arbor are preserved. This method is applicable across a variety of ganglion cell subtypes and is amenable to a wide variety of single-cell physiological techniques.

This article provides a description of how to dissect and record from the isolated retinal preparation in mouse. In particular, we describe how to record light responses from a fluorescently labeled ganglion cell population and subsequently identify and analyze its morphology.

Una preparación aislada de retina para registrar la respuesta de la luz a partir de semillas etiquetadas las células ganglionares retinianas

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1.  This information is then ...

Investigación

Neurociencias

Protocol for Three-dimensional Confocal Morphometric Analysis of Astrocytes

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify their functional and structural properties, a condition called reactive astrogliosis. Here, a protocol for assessment of the morphological properties of astrocytes is presented. This protocol includes quantification of 12 different parameters i.e. the surface area and volume of the tissue covered by an astrocyte (astrocyte territory), the entire astrocyte including branches, cell body, and nucleus, as well as total length and number of branches, the intensity of fluorescence immunoreactivity of antibodies used for astrocyte detection, and astrocyte density (number/1,000 &#181;m2). For this purpose three-dimensional (3D) confocal microscopic images were created, and 3D image analysis software such as Volocity 6.3 was used for measurements. Rat brain tissue exposed to amyloid beta1-40 (A&#946;1-40) with or without a therapeutic&#160;intervention was used to present the method. This protocol can also be used for 3D morphometric analysis of other cells from either in vivo or in vitro conditions.

Astrocytes in the CNS change their functional and structural properties in response to harmful stimuli. This report presents a protocol for assessment of three-dimensional astrocyte morphology in diseased conditions or after therapeutic interventions.

Protocolo para tridimensional confocal morfométrico Análisis de astrocitos

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify ...

Immunostaining of Biocytin-filled and Processed Sections for Neurochemical Markers

Electrophysiological recordings of cells using the patch clamp technique have allowed for the identification of different neuronal types based on firing patterns. The inclusion of biocytin/neurobiotin in the recording electrode permits post-hoc recovery of morphological details, which are necessary to determine the dendritic arborization and the regions targeted by the axons of the recorded neurons. However, given the presence of morphologically similar neurons with distinct neurochemical identities and functions, immunohistochemical staining for cell-type-specific proteins is essential to definitively identify neurons. To maintain network connectivity, brain sections for physiological recordings are prepared at a thickness of 300 &#181;m or greater. However, this thickness often hinders immunohistological postprocessing due to issues with antibody penetration, necessitating the resectioning of the tissue. Resectioning of slices is a challenging art, often resulting in the loss of tissue and morphology of the cells from which electrophysiological data was obtained, rendering the data unusable. Since recovery of morphology would limit data loss and guide in the selection of neuronal markers, we have adopted a strategy of recovering cell morphology first, followed by secondary immunostaining. We introduce a practical approach to biocytin filling during physiological recordings and subsequent serial immunostaining for the recovery of morphology, followed by the restaining of sections to determine the neurochemical identity. We report that sections that were filled with biocytin, fixed with paraformaldehyde (PFA), stained, and coverslipped can be removed and restained with a second primary antibody days later. This restaining involves the removal of the coverslip, the washing of sections in a buffer solution, and the incubation of primary and secondary antibodies to reveal the neurochemical identity. The method is advantageous for eliminating data loss due to an inability to recover morphology and for narrowing down the neurochemical markers to be tested based on morphology.

This protocol presents a method for the morphological recovery of neurons patched during electrophysiological recordings using biocytin filling and subsequent immunohistochemical postprocessing. We show that thick biocytin-filled sections that were stained and coverslipped can be restained with a second primary antibody days&#160;or months later.

La inmunotinción de las secciones llenas de Biocitina y se procesaron para neuroquímicos Marcadores

Electrophysiological recordings of cells using the patch clamp technique have allowed for the identification of different neuronal types based on firing ...

Microstate and Omega Complexity Analyses of the Resting-state Electroencephalography

Microstate and omega complexity are two reference-free electroencephalography (EEG) measures that can represent the temporal and spatial complexities of EEG data and have been widely used to investigate the neural mechanisms in some brain disorders. The goal of this article is to describe the protocol underlying EEG microstate and omega complexity analyses step by step. The main advantage of these two measures is that they could eliminate the reference-dependent problem inherent to traditional spectrum analysis. In addition, microstate analysis makes good use of high time resolution of resting-state EEG, and the four obtained microstate classes could match the corresponding resting-state networks respectively. The omega complexity characterizes the spatial complexity of the whole brain or specific brain regions, which has obvious advantage compared with traditional complexity measures focusing on the signal complexity in a single channel. These two EEG measures could complement each other to investigate the brain complexity from the temporal and spatial domain respectively.

This article describes the protocol underlying electroencephalography (EEG) microstate analysis and omega complexity analysis, which are two reference-free EEG measures and highly valuable to explore the neural mechanisms of brain disorders.

Según los Estados y análisis de complejidad de Omega de la electroencefalografía de estado descansando

Microstate and omega complexity are two reference-free electroencephalography (EEG) measures that can represent the temporal and spatial complexities of ...

Preparation of Mouse Retinal Cryo-sections for Immunohistochemistry

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for determining the mechanisms underlying retinal diseases. Maintaining structural integrity throughout the tissue preparation is vital for obtaining reproducible retinal IHC data but can be challenging due to the fragility and complexity of retinal cytoarchitecture. Strong fixatives like 10% formalin or Bouin's solution optimally preserve the retinal structure, they often impede IHC analysis by enhancing the background fluorescence and/or diminishing antibody-epitope interactions, a process known as epitope masking. Milder fixatives, on the other hand, like 4% paraformaldehyde, reduces background fluorescence and epitope-masking, meticulous dissection techniques must be utilized to preserve the retinal structure. In this article, we present a comprehensive method to prepare mouse ocular posterior cups for IHC that is sufficient to preserve most antibody-epitope interactions without loss of retinal structural integrity. We include representative IHC with antibodies to various retinal cell type markers to illustrate tissue preservation and orientation under optimal and sub-optimal conditions. Our goal is to optimize IHC studies of the retina by providing a complete protocol from ocular posterior cup dissection to IHC.

This report describes comprehensive methods for preparing frozen mouse retina sections for immunohistochemistry (IHC). Methods described include dissection of the ocular posterior cup, paraformaldehyde fixation, embedding in Optimal Cutting Temperature (OCT) media and tissue orientation, sectioning and immunostaining.

Preparación de criosecciones retinianas de ratón para inmunohistoquímica

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for ...

Preparation of Newborn Rat Brain Tissue for Ultrastructural Morphometric Analysis of Synaptic Vesicle Distribution at Nerve Terminals

Our laboratory and many others have exploited the high resolving power of transmission electron microscopy to study the morphology and spatial organization of synaptic vesicles. In order to obtain high-quality electron micrographs that can yield the degree of morphological detail necessary for quantitative analysis of pre-synaptic vesicle distribution, optimal specimen preparation is critical. Chemical fixation is the first step in the process of specimen preparation, and of utmost importance to preserve fine ultrastructure. Vascular fixation with a glutaraldehyde-formaldehyde solution, followed by treatment of vibratome-sectioned specimens with osmium tetroxide, stabilizes the maximum number of molecules, especially proteins and lipids, and results in superior conservation of ultrastructure. Tissue is then processed with counterstaining, sequential dehydration and resin-embedding. En bloc staining with uranyl acetate (i.e., staining of vibratome-sectioned tissue before resin embedding) enhances endogenous contrast and stabilizes cell components against extraction during specimen processing. Contrast can be further increased by applying uranyl acetate as a post-stain on ultrathin sections. Double-staining of ultrathin sections with lead citrate after uranyl acetate treatment also improves image resolution, by intensifying electron-opacity of nucleic acid-containing structures through selective binding of lead to uranyl acetate. Transmission electron microscopy is a powerful tool for characterization of the morphological details of synaptic vesicles and quantification of their size and spatial organization in the terminal bouton. However, because it uses fixed tissue, transmission electron microscopy can only provide indirect information regarding living or evolving processes. Therefore, other techniques should be considered when the main objective is to study dynamic or functional aspects of synaptic vesicle trafficking and exocytosis.

We describe procedures for processing newborn rat brain tissue to obtain high-resolution electron micrographs for morphometric analysis of synaptic vesicle distribution at nerve terminals. The micrographs obtained with these methods can also be used to study the morphology of a number of other cellular components and their dimensional structural relationships.

Preparación del tejido cerebral de rata recién nacida para el análisis Morfolométrico ultraestructural de la distribución de vesículas sinápticas en los terminales nerviosos

Our laboratory and many others have exploited the high resolving power of transmission electron microscopy to study the morphology and spatial ...

Collection of Frozen Rodent Brain Regions for Downstream Analyses

As our understanding of neurobiology has progressed, molecular analyses are often performed on small brain areas such as the medial prefrontal cortex (mPFC) or nucleus accumbens. The challenge in this work is to dissect the correct area while preserving the microenvironment to be examined. In this paper, we describe a simple, low-cost method using resources readily available in most labs. This method preserves nucleic acid and proteins by keeping the tissue frozen throughout the process. Brains are cut into 0.5&#8211;1.0 mm sections using a brain matrix and arranged on a frozen glass plate. Landmarks within each section are compared to a reference, such as the Allen Mouse Brain Atlas, and regions are dissected using a cold scalpel or biopsy punch. Tissue is then stored at -80 &#176;C until use. Through this process rat and mouse mPFC, nucleus accumbens, dorsal and ventral hippocampus and other regions have been successfully analyzed using qRT-PCR and Western assays. This method is limited to brain regions that can be identified by clear landmarks.

This procedure describes the collection of discrete frozen brain regions to obtain high-quality protein and RNA using inexpensive and commonly available tools.

Colección de regiones cerebrales de roedores congelados para análisis posteriores

As our understanding of neurobiology has progressed, molecular analyses are often performed on small brain areas such as the medial prefrontal cortex ...

Preparation of the Rat Vocal Fold for Neuromuscular Analyses

The purpose of this tutorial is to describe the preparation of the rat vocal fold for histochemical neuromuscular study. This protocol outlines procedures for rat laryngeal dissection, flash-freezing, and cryosectioning of the vocal folds. This study describes how to cryosection vocal folds in both longitudinal and cross-sectional planes. A novelty of this protocol is the laryngeal tracking during cryosectioning that ensures accurate identification of the intrinsic laryngeal muscles and reduces the chance of tissue loss. Figures demonstrate the progressive cryosectioning in both planes. Twenty-nine rat hemi-larynges were cryosectioned and tracked from the emergence of the thyroid cartilage to the appearance of the first section that included the full vocal fold. The full vocal fold was visualized for all animals in both planes. There was high variability in the distance from the appearance of the thyroid cartilage to the appearance of the full vocal fold in both planes. Weight was not correlated to depth of laryngeal landmarks, suggesting individual variability and other factors related to tissue preparation may be responsible for the high variability in the appearance of landmarks during sectioning. This study details a methodology and presents morphological data for preparing the rat vocal fold for histochemical neuromuscular investigation. Due to high individual variability, laryngeal landmarks should be closely tracked during cryosectioning to prevent oversectioning tissue and tissue loss. The use of a consistent methodology, including adequate tissue preparation and awareness of landmarks within the rat larynx, will assist with consistent results across studies and aid new researchers interested in using the rat vocal fold as a model to investigate laryngeal neuromuscular mechanisms.

This protocol describes methods used to prepare rat vocal folds for histochemical neuromuscular study.

Preparación de la cuerda vocal de la rata para análisis neuromusculares

The purpose of this tutorial is to describe the preparation of the rat vocal fold for histochemical neuromuscular study. This protocol outlines procedures ...

Dissection of Single Skeletal Muscle Fibers for Immunofluorescent and Morphometric Analyses of Whole-Mount Neuromuscular Junctions

The neuromuscular junction (NMJ) is a specialized point of contact between the motor nerve and the skeletal muscle. This peripheral synapse exhibits high morphological and functional plasticity. In numerous nervous system disorders, NMJ is an early pathological target resulting in neurotransmission failure, weakness, atrophy, and even in muscle fiber death. Due to its relevance, the possibility to quantitatively assess certain aspects of the relationship between NMJ components can help to understand the processes associated with its assembly/disassembly. The first obstacle when working with muscles is to gain the technical expertise to quickly identify and dissect without damaging their fibers. The second challenge is to utilize high-quality detection methods to obtain NMJ images that can be used to perform quantitative analysis. This article presents a step-by-step protocol for dissecting extensor digitorum longus and soleus muscles from rats. It also explains the use of immunofluorescence to visualize pre and postsynaptic elements of whole-mount NMJs. Results obtained demonstrate that this technique can be used to establish the microscopic anatomy of the synapsis and identify subtle changes in the status of some of its components under physiological or pathological conditions.

The ability to accurately detect neuromuscular junction components is crucial in evaluating modifications in its architecture because of pathological or developmental processes. Here we present a complete description of a straightforward method to obtain high-quality images of whole-mount neuromuscular junctions that can be used to perform quantitative measurements.

Disección De Fibras Musculares Esqueléticas Individuales Para Análisis Inmunofluorescentes Y Morfométricos De Las Uniones Neuromusculares De Montaje Completo

The neuromuscular junction (NMJ) is a specialized point of contact between the motor nerve and the skeletal muscle. This peripheral synapse exhibits high ...

Quantification of Immunostained Caspase-9 in Retinal Tissue

The family of caspases is known to mediate many cellular pathways beyond cell death, including cell differentiation, axonal pathfinding, and proliferation. Since the identification of the family of cell death proteases, there has been a search for tools to identify and expand the function of specific family members in development, health, and disease states. However, many of the currently commercially available caspase tools that are widely used are not specific for the targeted caspase. In this report, we delineate the approach we have used to identify, validate, and target caspase-9 in the nervous system using a novel inhibitor and genetic approaches with immunohistochemical read-outs. Specifically, we used the retinal neuronal tissue as a model to identify and validate the presence and function of caspases. This approach enables the interrogation of cell-type specific apoptotic and non-apoptotic caspase-9 functions and can be applied to other complex tissues and caspases of interest. Understanding the functions of caspases can help to expand current knowledge in cell biology, and can also be advantageous to identify potential therapeutic targets due to their involvement in disease.

Presented here is a detailed immunohistochemistry protocol to identify, validate, and target functionally relevant caspases in complex tissues.