O trabalho de pesquisa olho neste laboratório é apoiado pela Fundação Americana de Saúde Adjunto, Azaléia (1972) Fundo de Doações, e do Fundo HKU Projeto Pequeno (20097176185).

1. Ferramentas Microscópio, Nikon Investigador Stereo, MBF Bioscience - MicroBrightField, Inc. 2. Preparação <ol><li> Antes de trabalhar em qualquer análise morfométrica, cada amostra de retina é seccionado em 4 mícrons de espessura e sofre a coloração H &amp; E. </li><li> A secção de retina é dividida em 4 regiões - região periférica superior, a região central superior, a parte inferior região periférica, e quanto menor região central. </li><li> Depois de definir as regiões, uma imagem de cada região é adquirida em ampliações 10x40 sob o microscópio feixe luminoso da Nikon. </li><li> Agora, estamos prontos para analisar a morfometria da retina. </li></ol> 3. Medição da espessura INL <ol><li> Quatro medições de espessura INL em cada região foram medidos. Quatro linhas serão desenhadas perpendicularmente a partir da base de contorno para a borda externa do INL. </li><li>Abra o software chamado Stereo Investigator e escolher a lente 40x da barra de ferramentas principal. </li><li> Selecione &quot;Abrir Imagem&quot; na guia &quot;Arquivo&quot;. Escolha a imagem de uma região desejada da retina que você gostaria de analisar. </li><li> Escolha o tipo de linha de contorno da caixa de lista suspensa da barra de ferramentas principal e pressione o botão &quot;Auto Move&quot; botão. </li><li> Botão direito do mouse na janela de rastreamento, e selecione &quot;Simple Clique Tracing&quot;. </li><li> Desenhe a linha de contorno ao longo da fronteira interna do INL. </li><li> Quando o desenho é feito, com o botão direito na janela de rastreamento e escolha &quot;Contour Open End&quot;. </li><li> Seleccionar um marcador na barra de ferramentas marcador e adicionar marcadores nos pontos de junção. </li><li> Depois de adicionar todos os marcadores, desmarcar o ícone de marcador na barra de ferramentas do marcador. </li><li> Posicionar um cursor no centro de um dos marcadores. </li></ol><img src="/files/ftp_upload/3377/3377fig1.jpg" alt="A Figura 1" /><ol start="11"><li> Selecione &quot;QuickMedir o ângulo &quot;na guia&quot; Ferramentas &quot;. </li></ol><img src="/files/ftp_upload/3377/3377fig2.jpg" alt="A Figura 2" /><ol start="12"><li> Botão direito do mouse na janela de rastreamento e desmarcar a opção &quot;Contínuo&quot;. </li><li> Medir um ângulo clicando primeiro no centro de um marcador, que é adjacente ao marcador seleccionado para uma extremidade do segmento de linha. </li></ol><img src="/files/ftp_upload/3377/3377fig3.jpg" alt="A Figura 3" /><ol start="14"><li> Em seguida, mova o mouse para o centro do marcador em questão e clique novamente. </li><li> Estender a linha de elástico para a borda externa do INL. </li><li> Ajustar o ângulo até 90 ° é atingido. </li></ol><img src="/files/ftp_upload/3377/3377fig4.jpg" alt="A Figura 4" /><ol start="17"><li> Clique deixado na posição desejada. Uma caixa de diálogo mostrando uma medição do ângulo irá aparecer logo após o clique. </li><li> Pressione o botãoTecla &quot;Enter&quot;, mantendo o rato ainda. </li></ol><img src="/files/ftp_upload/3377/3377fig5.jpg" alt="A Figura 5" /><ol start="19"><li> Clique esquerdo da janela de rastreio de modo que uma linha é traçada a partir de uma extremidade à extremidade novo seleccionada. </li><li> Botão direito do mouse na janela de rastreamento e escolha &quot;Contour Open End&quot;. </li></ol><img src="/files/ftp_upload/3377/3377fig6.jpg" alt="A Figura 6" /><ol start="21"><li> Desenhar as linhas outros em diferentes posições no exactamente da mesma maneira como descrito anteriormente. </li><li> Depois de desenhar todas as linhas, selecione o modo de edição e renomear os 4 segmentos de linha em conformidade. </li><li> Quatro medidas podem ser obtidas pressionando &quot;Medição Contour&quot; botão. </li><li> Selecione essas medidas 4 e colá-los para uma planilha, clicando em &quot;Copiar para a Área de Transferência&quot;. </li><li> Selecione &quot;Salvar como&quot; na aba &quot;Arquivo&quot;. Os dados de rastreamento pode ser salva como um arquivo de dados. </li></ol><ol><li> Selecione &quot;Abrir Imagem&quot; na guia &quot;Arquivo&quot;. Escolha a imagem de uma região desejada da retina que você gostaria de analisar. </li><li> Escolha o tipo de linha de contorno da caixa de lista suspensa da barra de ferramentas principal. </li><li> Desenhar uma linha de contorno ao longo da fronteira da camada de fibras nervosas (NFL). </li><li> Termine o rastreamento com o &quot;Contour Open End&quot; opção. </li><li> Em cada extremidade, adicionar um marcador na ponta da linha e outro na posição perto da ponta. </li><li> Desmarque o ícone marcador na barra de ferramentas do marcador. </li><li> Posicionar um cursor no centro dos marcadores na ponta. </li><li> Selecione &quot;Angle medida rápida&quot; na guia &quot;Ferramentas&quot;. </li><li> Botão direito do mouse na janela de rastreamento e desmarcar a opção &quot;Contínuo&quot;. </li><li> Ajustar as linhas da faixa de borracha e medir um ângulo de 90 °. </li><li> Pressione a tecla &quot;Enter&quot; depois de uma caixa de diálogo é apareceu. </li><li> Esquerda clique em tele rastreio janela de modo que uma nova linha pode ser estendida perpendicularmente a partir da ponta. </li><li> Desenhe outra linha nova no exatamente da mesma maneira no outro fim. </li><li> Dois limites são criados dentro do qual o número de RGCs é contado. </li><li> Escolha uma fronteira como uma linha de exclusão que os toques de células não é contado. </li><li> Selecione um tipo de marcador e anexar um para cada célula viva. </li><li> Selecione outro tipo de marcador para as células mortas. </li><li> O número de cada tipo de marcadores colocados será exibido com o ícone marcador em conformidade na barra de ferramentas marcador. </li><li> Selecione o modo de edição e pegar a linha de base de contorno. Botão direito do mouse na janela de rastreamento e renomear a linha. </li><li> O comprimento da linha de base contorno pode ser obtido por compressão de &quot;medição de contorno&quot; botão. </li><li> O número de células podem então ser expresso por mm de comprimento. </li><li> Selecione &quot;Salvar como&quot; na aba &quot;Arquivo&quot;. Orastreamento e os dados de marcadores podem ser salvos como um arquivo de dados. </li></ol> 5. Medindo o tamanho da célula de uma célula RGC <ol><li> Selecione &quot;Abrir Imagem&quot; na guia &quot;Arquivo&quot;. Escolha a imagem de uma região desejada da retina que você gostaria de analisar. </li><li> Zoom em uma célula viva e escolha o tipo de linha de contorno da caixa de lista suspensa da barra de ferramentas principal. </li><li> Desenhar uma linha de contorno ao longo da circunferência da célula. </li><li> Por volta do fim, com o botão direito na janela de rastreamento e selecione &quot;End contorno próximo&quot;. </li><li> A área da célula será exibido no item de Área na caixa de diálogo Contorno Medição. </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>

Os autores não têm nada a revelar.

1. Como obter uma medição de espessura mais precisa? Você pode ampliar a imagem clicando em &quot;Zoom in&quot; e à esquerda, clique na janela de rastreamento. A fronteira INL pode ser visto claramente. Se a ponta da linha não está na borda externa do INL, podemos ajustar a linha sob o modo de edição. Após escolher a linha em causa, com o botão direito na janela de rastreamento e escolha &quot;Ponto de Inserção na Contour Selecionado&quot;. Um ponto pode ser adicionado na fronteira ...

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> Nome do reagente </td><td> Tipo </td><td> Companhia </td><td> Número de catálogo </td></tr><tr><td> Investigador Stereo </td><td> MBF Software Análise Bioscience </td><td> Campo MicroBright </td><td></td></tr><tr><td> Microscópio </td><td> Olympus BX51 </td><td> Olympus Corporation </td><td> BX51 </td></tr></tbody></table>

morphometric analyses of retinal sections

Análises morfométricas de secções da retina têm sido utilizados na análise doenças da retina. Para exemplos, as células neuronais foram significativamente perdido na camada de células ganglionares da retina (RGCL) em modelos de ratos com N-metil-D-aspartato (NMDA)-induzida excitotoxicidade 1, lesão de isquemia-reperfusão da retina e glaucoma 2 3. Redução do INL e interior camada plexiforme (IPL) espessuras foram invertidos com o tratamento com citicolina em olhos de ratos submetidos a excitotoxicidade do glutamato mediada pelo ácido caínico 4. Alteração da densidade de RGC e tamanhos do soma foram observadas com tratamentos medicamentosos diferentes em olhos com pressão intra-ocular elevada 3,5,6. Portanto, ter métodos objetivos de analisar as morphometries na retina podem ser de grande importância na avaliação de patologias da retina e da eficácia de estratégias terapêuticas. A estrutura da retina é multi-camadas e vários tipos diferentes de neurônios exir na retina. Os parâmetros morfométricos do retina tais como o número de células, o tamanho da célula e da espessura das camadas de diferentes são mais complexas do que o sistema de cultura celular. Logo no início, estes parâmetros podem ser detectadas usando software de imagem de outro comercial. Os valores são normalmente de valor relativo, e mudando para o valor exacto pode necessitar de cálculo mais preciso. Além disso, o traçado do tamanho da célula e morfologia pode não ser preciso e suficientemente sensível para a análise estatística, especialmente no modelo de glaucoma crónico. As medições utilizados neste protocolo fornecido um modo mais preciso e rápido. E o comprimento absoluto da linha e tamanho da célula pode ser directa e fácil de ser copiado para outros arquivos. Por exemplo, descrevemos a história da margem de núcleos mais interior e exterior, no INL e formou uma linha, em seguida, usando o software para desenhar um ângulo de 90 graus para medir a espessura. Embora sem a ajuda do software, a linha oblíqua e talvez a mudança de espessura da retina podenão ser repetitivo entre observadores individuais. Além disso, o número e densidade de RGCs também pode ser quantificada. Este protocolo com sucesso diminui a variabilidade na quantificação características da retina, aumenta a sensibilidade na detecção de alterações mínimas. Este vídeo vai demonstrar três tipos de análises morfométricas das secções da retina. Eles incluem a medição da espessura INL, quantificar o número de RGCs e medição dos tamanhos de RGCs em valor absoluto. Estas três análises são realizadas com Stereo Investigator (MBF Bioscience - MicroBrightField, Inc.). A técnica pode oferecer uma plataforma simples, mas científica para análises morfométricas.

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

Morphometric Analyses of Retinal Sections

Este vídeo demonstra três tipos de análise morfométrica da retina, que incluem a medição da espessura da camada nuclear interna, a quantificação do número de células ganglionares da retina (RGCs) e medindo os tamanhos dos RGCs. A técnica pode oferecer uma plataforma simples, mas científica para análises morfométricas.

Análises morfométricas de secções da 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 visualizações.  The University of Hong Kong. Este vídeo demonstra três tipos de análise morfométrica da retina, que incluem a medição da espessura da camada nuclear interna, a quantificação do número de células ganglionares da retina (RGCs) e medindo os tamanhos dos RGCs. A técnica pode oferecer uma plataforma simples, mas científica para análises morfométricas.

Vídeo: 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.

Uma preparação isolada da retina para gravar Resposta Luz geneticamente identificadas células ganglionares da retina

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

Neurociência

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 morfométrica Análise de astrócitos

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.

Imunocoramento Secções biocitina-preenchidos e processados ​​para neuroquímicos Markers

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.

Microestado e análises de complexidade Omega de eletroencefalografia a 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.

Preparação de Cryo-seções retinal do rato para immunohistochemistry

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.

Preparação do tecido cerebral de rato recém-nascido para Análise morfométrica ultraestrutural da distribuição de vesículas sinápticas em terminais nervosos

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.

Coleção de regiões cerebrais de roedores congelados para análises a jusante

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.

Preparação da Dobra Vocal do Rato para Análises 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.

Dissecção de fibras musculares esqueléticas únicas para análises imunofluorescentes e morfométricas de junções neuromusculares de monte inteiro

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.