Le travail sur la recherche des yeux dans ce laboratoire est soutenu par la Fondation panaméricaine de la santé adjoint, Azalée (1972) Fonds de dotation, et le Fonds HKU des petits projets (20097176185).

1. Outils Microscope, Nikon Enquêteur stéréo, MBF Bioscience - MicroBrightField, Inc 2. Préparation <ol><li> Avant de travailler sur une analyse morphométrique, chaque échantillon rétine est sectionné à 4 microns d&#39;épaisseur et subit la coloration H &amp; E. </li><li> La section rétine est divisée en 4 régions - la région supérieure périphérique, la région centrale supérieure, la région périphérique inférieure, et la région inférieure centrale. </li><li> Après avoir défini les régions, une image de chaque région est acquise à des grossissements 10x40 sous le microscope faisceau lumineux de Nikon. </li><li> Maintenant, nous sommes prêts à analyser la morphométrie de la rétine. </li></ol> 3. Mesure de l&#39;épaisseur INL <ol><li> Quatre mesures de l&#39;épaisseur INL dans chaque région ont été mesurés. Quatre lignes sera tirée perpendiculairement à partir de la ligne de base de contour de la frontière extérieure de la INL. </li><li>Ouvrez le logiciel appelé chercheur stéréo et choisissez la lentille 40x de la barre d&#39;outils principale. </li><li> Sélectionnez &quot;Ouvrir une image&quot; sous l&#39;onglet «Fichier». Choisissez une image d&#39;une région désirée rétine que vous souhaitez analyser. </li><li> Choisissez le type de ligne de contour de la zone de liste déroulante de la barre d&#39;outils principale et appuyez sur le &quot;Move Auto&quot;. </li><li> Faites un clic droit dans la fenêtre de traçage, et sélectionnez &quot;Simple Click traçage&quot;. </li><li> Dessiner la ligne de contour le long de la bordure intérieure de l&#39;INL. </li><li> Lorsque le dessin est fait, faites un clic droit dans la fenêtre de traçage et de choisir &quot;Contour Open End&quot;. </li><li> Sélectionnez un marqueur à la barre d&#39;outils marqueur et ajouter des marqueurs aux points de jonction. </li><li> Après l&#39;ajout de tous les marqueurs, désélectionner l&#39;icône marqueur à la barre d&#39;outils marqueurs. </li><li> Positionner un curseur au centre de l&#39;un des marqueurs. </li></ol><img src="/files/ftp_upload/3377/3377fig1.jpg" alt="Figure 1" /><ol start="11"><li> Sélectionnez «QuickMesurer l&#39;angle &quot;sous l&#39;onglet&quot; Outils &quot;. </li></ol><img src="/files/ftp_upload/3377/3377fig2.jpg" alt="Figure 2" /><ol start="12"><li> Faites un clic droit dans la fenêtre de traçage et de désélectionner le &quot;continu&quot; option. </li><li> Mesurer un angle en cliquant d&#39;abord au centre d&#39;un marqueur, qui est adjacente au marqueur sélectionné pour une extrémité du segment de ligne. </li></ol><img src="/files/ftp_upload/3377/3377fig3.jpg" alt="Figure 3" /><ol start="14"><li> Puis, déplacez la souris vers le centre du marqueur concerné et cliquez à nouveau. </li><li> Prolonger la ligne bande de caoutchouc de la frontière extérieure de l&#39;INL. </li><li> Ajustez l&#39;angle jusqu&#39;à 90 ° est atteint. </li></ol><img src="/files/ftp_upload/3377/3377fig4.jpg" alt="Figure 4" /><ol start="17"><li> Clic gauche à la position désirée. Une boîte de dialogue affichant une mesure d&#39;angle apparaîtra juste après le clic. </li><li> Appuyez sur la toucheTouche &quot;Enter&quot; tout en maintenant la souris. </li></ol><img src="/files/ftp_upload/3377/3377fig5.jpg" alt="Figure 5" /><ol start="19"><li> Un clic gauche sur la fenêtre de traçage de sorte qu&#39;une ligne est tracée d&#39;un bout à la fin nouveau sélectionné. </li><li> Faites un clic droit dans la fenêtre de traçage et de choisir &quot;Contour Open End&quot;. </li></ol><img src="/files/ftp_upload/3377/3377fig6.jpg" alt="Figure 6" /><ol start="21"><li> Tracer les lignes d&#39;autres à des positions différentes, exactement de la même manière que décrit précédemment. </li><li> Après avoir dessiné toutes les lignes, sélectionnez le mode d&#39;édition et renommer les 4 segments de ligne en conséquence. </li><li> Quatre mesures peuvent être obtenues en appuyant sur &quot;Mesure Contour&quot; bouton. </li><li> Sélectionnez ces 4 mesures et de les coller dans une feuille de calcul en cliquant sur &quot;Copy to Clipboard&quot;. </li><li> Sélectionnez «Enregistrer sous» sous l&#39;onglet «Fichier». Les données de suivi peuvent être sauvegardés comme un fichier de données. </li></ol><ol><li> Sélectionnez &quot;Ouvrir une image&quot; sous l&#39;onglet «Fichier». Choisissez une image d&#39;une région désirée rétine que vous souhaitez analyser. </li><li> Choisissez le type de ligne de contour de la zone de liste déroulante de la barre d&#39;outils principale. </li><li> Dessinez une ligne de contour le long de la frontière de la couche de fibres nerveuses (NFL). </li><li> Mettre fin à la traçabilité avec le &quot;Contour Open End&quot; option. </li><li> À chaque extrémité, ajouter un marqueur à l&#39;extrémité de la ligne et une autre à la position proche de la pointe. </li><li> Désélectionnez l&#39;icône marqueur à la barre d&#39;outils marqueur. </li><li> Positionner un curseur au centre des marqueurs à la pointe. </li><li> Sélectionnez «Angle Mesure rapide» sous l&#39;onglet «Outils». </li><li> Faites un clic droit dans la fenêtre de traçage et de désélectionner le &quot;continu&quot; option. </li><li> Ajuster les lignes de bande en caoutchouc et mesurer un angle de 90 °. </li><li> Appuyez sur la touche &quot;Entrée&quot; après une boîte de dialogue est sorti. </li><li> Clic gauche til traçage fenêtre de sorte qu&#39;une nouvelle ligne peut être étendue perpendiculairement à partir de la pointe. </li><li> Tracez une autre ligne nouvelle dans le exactement de la même façon à l&#39;autre extrémité. </li><li> Deux limites sont créés dans lequel le nombre de RGC est compté. </li><li> Choisissez une frontière comme une ligne d&#39;exclusion qui touche les cellules ne sont pas comptés. </li><li> Sélectionnez un type de marqueur et joindre une à chaque cellule vivante. </li><li> Sélectionnez un autre type de marqueur pour les cellules mortes. </li><li> Le nombre de chaque type de marqueurs placés seront affichés à côté de l&#39;icône marqueur en conséquence à la barre d&#39;outils marqueur. </li><li> Sélectionnez le mode d&#39;édition et de ramasser la base du contour. Faites un clic droit dans la fenêtre de traçage et de renommer la ligne. </li><li> La longueur de la ligne de base du contour peut être obtenu en appuyant sur &quot;Mesure Contour&quot; bouton. </li><li> Le nombre des cellules peut alors être exprimé par mm de la longueur. </li><li> Sélectionnez «Enregistrer sous» sous l&#39;onglet «Fichier». Lale traçage et les données de marqueurs peut être sauvegardé comme un fichier de données. </li></ol> 5. Mesurer la taille de cellule d&#39;une cellule RGC <ol><li> Sélectionnez &quot;Ouvrir une image&quot; sous l&#39;onglet «Fichier». Choisissez une image d&#39;une région désirée rétine que vous souhaitez analyser. </li><li> Zoom dans une cellule vivante et choisissez le type de ligne de contour de la zone de liste déroulante de la barre d&#39;outils principale. </li><li> Tracer une ligne de contour le long de la circonférence de la cellule. </li><li> Vers la fin, faites un clic droit de la fenêtre de traçage et de sélectionner &quot;contour Fin proche&quot;. </li><li> La surface de la cellule sera affiché sous la rubrique de la zone dans la boîte de dialogue Contour mesure. </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>

Les auteurs n&#39;ont rien à révéler.

1. Comment faire pour obtenir une mesure d&#39;épaisseur plus précis? Vous pouvez agrandir l&#39;image en cliquant sur &quot;Zoom&quot; et faites un clic gauche dans la fenêtre de traçage. La frontière INL peut être vu clairement. Si la pointe de la ligne n&#39;est pas à la frontière extérieure de l&#39;INL, nous pouvons ajuster la ligne sous le mode d&#39;édition. Après avoir ramassé la ligne concernée, faites un clic droit de la fenêtre de traçage et de choisir &quot;Insérer u...

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> Nom du réactif </td><td> Type </td><td> Entreprise </td><td> Numéro de catalogue </td></tr><tr><td> Enquêteur stéréo </td><td> Logiciel d&#39;analyse MBF Bioscience </td><td> Champ MicroBright </td><td></td></tr><tr><td> Microscope </td><td> Olympus BX51 </td><td> Olympus Corporation </td><td> BX51 </td></tr></tbody></table>

morphometric analyses of retinal sections

Les analyses morphométriques de sections rétiniennes ont été utilisés dans l&#39;examen de maladies de la rétine. Pour des exemples, des cellules neuronales ont été considérablement perdu dans la couche de cellules ganglionnaires de la rétine (RGCL) chez le rat avec la N-méthyl-D-aspartate (NMDA) excitotoxicité induite par 1, la rétine d&#39;ischémie-reperfusion 2 et le glaucome 3. Réduction de la couche plexiforme INL et interne (IPL) épaisseurs ont été inversés avec le traitement citicoline dans les yeux des rats soumis à l&#39;excitotoxicité du glutamate acide kaïnique médiée par 4. Modification de la densité et la taille des soma RGC ont été observés avec différents traitements médicamenteux dans les yeux avec une pression intraoculaire élevée 3,5,6. Par conséquent, ayant des méthodes objectives de l&#39;analyse des morphometries rétiniennes peuvent être d&#39;une grande importance dans l&#39;évaluation des pathologies de la rétine et l&#39;efficacité des stratégies thérapeutiques. La structure de la rétine est multi-couches et plusieurs différents types de neurones exier dans la rétine. Les paramètres morphométriques de la rétine comme le nombre de cellules, la taille des cellules et l&#39;épaisseur des différentes couches sont plus complexes que le système de culture cellulaire. Dès le début, ces paramètres peuvent être détectés à l&#39;aide d&#39;autres logiciels d&#39;imagerie commerciale. Les valeurs sont normalement de la valeur relative, et le passage à la valeur précise peut-être besoin de calcul plus précis. En outre, le tracé de la taille des cellules et la morphologie peuvent ne pas être exacts et suffisamment sensible pour l&#39;analyse statistique, en particulier dans le modèle de glaucome chronique. Les mesures utilisées dans le présent protocole a fourni un moyen plus précis et facile. Et la longueur absolue de la ligne et la taille de la cellule peut être signalé directement et facile à copier d&#39;autres fichiers. Par exemple, nous avons retracé la marge de la plupart des noyaux interne et externe dans l&#39;INL et ont formé une ligne, puis en utilisant le logiciel pour dessiner un angle de 90 degrés pour mesurer l&#39;épaisseur. Bien que sans l&#39;aide du logiciel, la ligne peut-être oblique et l&#39;évolution de l&#39;épaisseur de la rétine peutne pas être reproductible parmi les observateurs individuels. En outre, le nombre et la densité de CGR peut également être quantifiée. Ce protocole diminue avec succès la variabilité dans la quantitation caractéristiques de la rétine, augmente la sensibilité dans la détection des changements minimes. Cette vidéo démontre trois types d&#39;analyses morphométriques des sections rétiniennes. Ils comprennent la mesure de l&#39;épaisseur INL, quantifier le nombre de CGR et de mesurer les tailles de CGR en valeur absolue. Ces trois analyses sont effectuées avec l&#39;enquêteur stéréo (MBF Bioscience - MicroBrightField, Inc.) La technique peut offrir une plate-forme simple mais scientifique pour analyses morphométriques.

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

Morphometric Analyses of Retinal Sections

Cette vidéo montre trois types d&#39;analyses morphométriques de la rétine, qui comprennent la mesure de l&#39;épaisseur de couche nucléaire interne, de quantifier le nombre de cellules ganglionnaires de la rétine (CGR) et de mesurer les tailles de CGR. La technique peut offrir une plate-forme simple mais scientifique pour analyses morphométriques.

Analyses morphométriques des sections rétiniennes

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

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Neuroscience

10.2K vues.  The University of Hong Kong. Cette vidéo montre trois types d'analyses morphométriques de la rétine, qui comprennent la mesure de l'épaisseur de couche nucléaire interne, de quantifier le nombre de cellules ganglionnaires de la rétine (CGR) et de mesurer les tailles de CGR. La technique peut offrir une plate-forme simple mais scientifique pour analyses morphométriques.

Vidéo: 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.

Une préparation isolée rétinienne pour enregistrer la réponse de Lumière génétiquement marqués cellules ganglionnaires rétiniennes

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

Recherche

Neurosciences

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.

Protocole pour tridimensionnelle confocale Analyse morphométrique des 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 ...

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.

Immunocoloration des articles biocytine remplis et transformés pour neurochimiques Marqueurs

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.

Micro-État et Analyses complexité Omega de l’état de repos l’électroencéphalographie

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.

Préparation de la souris Retinal Cryo-sections pour l'immunohistochimie

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.

Préparation du tissu cérébral nouveau-né du rat pour l’analyse morphométrique ultrastructurale de la distribution synaptique des vésicules aux terminaux nerveux

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.

Collection de régions de cerveau de rongeur congelés pour des analyses en aval

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.

Préparation du pli vocal du rat pour les analyses neuromusculaires

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.

Dissection des fibres musculaires squelettiques simples pour les analyses immunofluorescentes et morphométriques des jonctions neuromusculaires de mont entier

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.