Die Arbeit am Auge Forschung in diesem Labor wird von der American Health Assistant-Stiftung, Azalee (1972) Endowment Fund, und HKU Small Project Fund (20097176185) unterstützt.

1. Werkzeuge Mikroskop, Nikon Stereo Investigator, MBF Bioscience - MicroBrightField, Inc. 2. Vorbereitung <ol><li> Vor Arbeiten an jedem morphometrische Analyse ist jeder Netzhaut Probe bei 4 Mikrometer Dicke geschnitten und unterzieht sich der H &amp; E Färbung. </li><li> Oberen Randbereich der oberen mittleren Bereich der unteren Randbereich, und die unteren mittleren Bereich - Die Netzhaut Abschnitt ist in 4 Bereiche aufgeteilt. </li><li> Nach der Definition der Regionen wird ein Bild der jeweiligen Region bei 10x40 Vergrößerungen unter dem hellen Lichtstrahl Mikroskop von Nikon erworben. </li><li> Nun sind wir bereit, Morphometrie der Netzhaut zu untersuchen. </li></ol> 3. Mess-INL Dicke <ol><li> Vier Messungen der INL Dicke in jeder Region wurden gemessen. Vier Linien werden senkrecht von der Kontur Basislinie gezogen werden, um den äußeren Rand des INL. </li><li>Öffnen Sie die Software mit dem Namen Stereo Investigator und wählen Sie das 40x Objektiv von der Haupt-Werkzeugleiste. </li><li> Wählen Sie &quot;Bild öffnen&quot; unter der Registerkarte &quot;Datei&quot;. Wählen Sie ein Bild von einem gewünschten retinalen Region, die Sie gerne analysieren würde. </li><li> Wählen Sie die Art der Konturlinie aus der Dropdown-Listenfeld der Haupt-Werkzeugleiste und drücken Sie die &quot;Auto Move&quot;-Taste. </li><li> Mitten im Tracing-Fenster klicken, und wählen Sie &quot;Simple Click-Tracing&quot;. </li><li> Zeichnen Sie die Konturlinie entlang der inneren Grenze des INL. </li><li> Wenn die Zeichnung fertig ist, direkt in der Tracing-Fenster und wählen Sie &quot;End Open Contour&quot;. </li><li> Wählen Sie einen Marker an der Marker Symbolleiste hinzufügen und Markierungen an den Verbindungsstellen. </li><li> Nach dem Hinzufügen alle Markierungen, deaktivieren Sie die Marker-Symbol in der Symbolleiste Marker. </li><li> Positionieren eines Cursors in der Mitte einer der Marker. </li></ol><img src="/files/ftp_upload/3377/3377fig1.jpg" alt="1" /><ol start="11"><li> Wählen Sie &quot;QuickMessen Sie Angle &quot;unter dem Reiter&quot; Tools &quot;. </li></ol><img src="/files/ftp_upload/3377/3377fig2.jpg" alt="2" /><ol start="12"><li> Mitten im Tracing-Fenster klicken und deaktivieren Sie die &quot;Continuous&quot;-Option. </li><li> Messen eines Winkels indem man zunächst in der Mitte einer Markierung, die benachbart zu der Markierung für ein Ende der Strecke ist, ausgewählt. </li></ol><img src="/files/ftp_upload/3377/3377fig3.jpg" alt="Abbildung 3" /><ol start="14"><li> Dann bewegen Sie die Maus in die Mitte des betreffenden Marker aus und klicken Sie erneut. </li><li> Erweitern Sie das Gummiband Linie an der äußeren Grenze des INL. </li><li> Stellen Sie den Winkel bis 90 ° erreicht wird. </li></ol><img src="/files/ftp_upload/3377/3377fig4.jpg" alt="Abbildung 4" /><ol start="17"><li> Linke Maustaste an die gewünschte Position. Ein Dialogfeld zeigt eine Winkelmessung öffnet sich direkt nach dem Klick. </li><li> Drücken Sie die Taste&quot;Enter&quot;-Taste bei gedrückter Maustaste immer noch. </li></ol><img src="/files/ftp_upload/3377/3377fig5.jpg" alt="Abbildung 5" /><ol start="19"><li> Linksklick auf den Tracing-Fenster, so dass eine Linie von einem Ende zum neuen ausgewählten Ende gezogen wird. </li><li> Mitten im Tracing-Fenster und wählen Sie &quot;Open-End Contour&quot;. </li></ol><img src="/files/ftp_upload/3377/3377fig6.jpg" alt="6" /><ol start="21"><li> Malen die anderen Linien an unterschiedlichen Positionen in genau der gleichen Weise wie zuvor beschrieben. </li><li> Nach dem Zeichnen Sie alle Zeilen, wählen Sie den Bearbeiten-Modus und benennen Sie die 4 Liniensegmenten entsprechend. </li><li> Vier Messungen können durch Drücken von &quot;Contour Measurement&quot;-Taste erreicht werden. </li><li> Wählen Sie diese 4 Messungen und fügen Sie sie in eine Tabelle, indem Sie auf &quot;Copy to Clipboard&quot;. </li><li> Wählen Sie &quot;Speichern unter&quot; unter der Registerkarte &quot;Datei&quot;. Die Tracing-Daten können als Datei gespeichert werden. </li></ol><ol><li> Wählen Sie &quot;Bild öffnen&quot; unter der Registerkarte &quot;Datei&quot;. Wählen Sie ein Bild von einem gewünschten retinalen Region, die Sie gerne analysieren würde. </li><li> Wählen Sie die Art der Konturlinie aus der Dropdown-Listenfeld auf der Hauptsymbolleiste. </li><li> Zeichnen Sie eine Konturlinie entlang der Grenze des Nervenfaserschicht (NFL). </li><li> Beenden Sie das Tracing mit der Option &quot;Open-End Contour&quot;. </li><li> An jedem Ende, fügen eine Markierung an der Spitze der Leitung und ein anderes an der Position nahe der Spitze. </li><li> Deaktivieren Sie die Marker-Symbol in der Symbolleiste Marker. </li><li> Positionieren Sie den Cursor in der Mitte der Markierungen an der Spitze. </li><li> Wählen Sie &quot;Quick-Measure Angle&quot; unter dem Reiter &quot;Tools&quot;. </li><li> Mitten im Tracing-Fenster klicken und deaktivieren Sie die &quot;Continuous&quot;-Option. </li><li> Passen Sie die Gummiband-Linien und messen Sie einen Winkel von 90 °. </li><li> Drücken Sie die &quot;Enter&quot;-Taste, nachdem ein Dialogfeld auftauchte. </li><li> Linksklick ter Tracing Fenster, so dass eine neue Linie kann senkrecht von der Spitze verlängert werden. </li><li> Zeichnen Sie eine weitere neue Zeile in der genau die gleiche Weise an einem anderen Ende. </li><li> Zwei Grenzen erzeugt werden, in dem die Anzahl von RGCs gezählt wird. </li><li> Wählen Sie eine Grenze als Ausschluss Linie, die die Zelle berührt nicht gezählt wird. </li><li> Wählen Sie eine Art von Markierung und fügen eine auf jeder lebenden Zelle. </li><li> Wählen Sie eine andere Art der Markierung für die toten Zellen. </li><li> Die Anzahl der einzelnen Arten von Markierungen platziert werden neben dem Marker-Symbol angezeigt werden dementsprechend an der Marker-Symbolleiste. </li><li> Wählen Sie den Bearbeitungsmodus und holen die Kontur Grundlinie. Mitten im Tracing-Fenster klicken und benennen Sie die Linie. </li><li> Die Länge der Grundlinie Kontur kann durch Drücken von &quot;Contour Measurement&quot;-Taste erreicht werden. </li><li> Die Anzahl der Zellen können dann pro mm der Länge ausgedrückt werden. </li><li> Wählen Sie &quot;Speichern unter&quot; unter der Registerkarte &quot;Datei&quot;. DieAufspüren und die Marker-Daten können als Datei gespeichert werden. </li></ol> 5. Messung der Zellgröße der Zellen RGC <ol><li> Wählen Sie &quot;Bild öffnen&quot; unter der Registerkarte &quot;Datei&quot;. Wählen Sie ein Bild von einem gewünschten retinalen Region, die Sie gerne analysieren würde. </li><li> Zoom in einer lebenden Zelle und wählen Sie den Typ der Konturlinie aus der Dropdown-Listenfeld auf der Hauptsymbolleiste. </li><li> Malen einer Konturlinie entlang des Umfangs der Zelle. </li><li> Gegen Ende der rechten Maustaste auf das Tracing-Fenster und wählen Sie &quot;End Kontur schließen&quot;. </li><li> Der Bereich der Zelle wird unter dem Punkt der Fläche in den Contour Measurement Dialogfeld angezeigt werden. </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>

Die Autoren haben nichts zu offenbaren.

1. Wie, um eine genauere Messung zu erhalten Dicke? Sie können das Bild durch Klicken auf &quot;Zoom in&quot;-Taste vergrößern und klicken Sie links im Fenster verfolgen. Der INL-Grenze ist deutlich zu erkennen. Wenn die Spitze der Linie ist nicht an der äußeren Grenze des INL, können wir die Zeile unter dem Bearbeiten-Modus einzustellen. Nach dem Pflücken werden die betreffenden Zeile der rechten Maustaste auf das Tracing-Fenster und wählen Sie &quot;Einfügen Point in ausgewählten Ko...

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> Name des Reagenzes </td><td> Typ </td><td> Firma </td><td> Katalog-Nummer </td></tr><tr><td> Stereo Investigator </td><td> MBF Bioscience Analyse-Software </td><td> MicroBright Feld </td><td></td></tr><tr><td> Mikroskop </td><td> Olympus BX51 </td><td> Olympus Corporation </td><td> BX51 </td></tr></tbody></table>

morphometric analyses of retinal sections

Morphometrische Analyse von Netzhautschnitte in Prüfung retinalen Krankheiten verwendet. Beispiele wurden neuronalen Zellen signifikant im retinalen Ganglienzellen (RGCL) im Rattenmodell mit N-Methyl-D-Aspartat verloren (NMDA) Exzitotoxizität 1, retinalen Ischämie-Reperfusionsverletzung 2 und 3-Glaukom induziert. Reduzierung der INL und inneren plexiformen Schicht (IPL) Dicken wurden mit Citicolin Behandlung bei Ratten &#39;Augen unterzogen Kainsäure-vermittelte Glutamatexzitotoxizität 4 umgekehrt. Änderung der RGC Dichte und Soma Größen wurden mit verschiedenen medikamentösen Behandlungen bei Augen mit erhöhtem Augeninnendruck 3,5,6 beobachtet. Daher kann mit objektiven Verfahren zur Analyse der retinalen morphometries von großer Bedeutung bei der Bewertung von retinalen Pathologien und die Wirksamkeit von therapeutischen Strategien sein. Das Retina-Struktur ist Multi-Schichten und verschiedene Arten von Neuronen Exist in der Netzhaut. Die morphometrischen Parameter der Retina, wie Zellzahl, Zellgröße und die Dicke der verschiedenen Schichten sind komplexer als der Zellkultur. Frühe auf, können diese Parameter unter Verwendung anderer kommerzieller Bildbearbeitungssoftware werden. Die Werte sind in der Regel von relativer Wert, und das Ändern der genaue Wert kann weiter eine genaue Berechnung müssen. Auch kann das Tracing der Zellgröße und Morphologie nicht genau und empfindlich genug für statistische Auswertung, vor allem in der chronischen Glaukom-Modell. Die Messungen in diesem Protokoll verwendet werden, sofern eine genauere und einfache Weise. Und die absolute Länge der Leitung und der Größe der Zelle kann direkt und einfach zu anderen Dateien kopiert werden gemeldet werden. Zum Beispiel verfolgt wir den Rand der inneren und äußeren meisten Kerne in der INL und eine Linie gebildet ist dann mit der Software, um eine 90-Grad-Winkel zu zeichnen, um die Dicke zu messen. Während ohne die Hilfe der Software kann die Leitung vielleicht schräg und personelle Veränderungen der Netzhautdickenicht wiederholbar sein zwischen den einzelnen Beobachtern. Darüber hinaus kann die Anzahl und Dichte von RGCs ebenfalls quantifiziert werden. Dieses Protokoll erfolgreich verringert die Variabilität in der quantitativen Merkmale der Netzhaut, erhöht die Empfindlichkeit bei der Erkennung minimale Änderungen. Dieses Video wird zeigen, drei Arten von morphometrischen Analysen der retinalen Abschnitten. Dazu gehören die Messung der Dicke INL, indem die Anzahl der RGCs und Messen der Größen von RGCs absolut. Diese drei Analysen werden mit Stereo-Investigator (- MicroBrightField, Inc. MBF Bioscience) durchgeführt. Die Technik bietet eine einfache, aber wissenschaftliche Plattform für morphometrische Analysen.

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

Morphometric Analyses of Retinal Sections

Dieses Video zeigt drei Arten von morphometrischen Analysen der Netzhaut, die Messung der inneren Körnerschicht Dicke, indem die Anzahl der retinalen Ganglienzellen (RGZ) und Messung der Größen von RGCs gehören. Die Technik bietet eine einfache, aber wissenschaftliche Plattform für morphometrische Analysen.

Morphometrische Analysen von Netzhautschnitte

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 Aufrufe.  The University of Hong Kong. Dieses Video zeigt drei Arten von morphometrischen Analysen der Netzhaut, die Messung der inneren Körnerschicht Dicke, indem die Anzahl der retinalen Ganglienzellen (RGZ) und Messung der Größen von RGCs gehören. Die Technik bietet eine einfache, aber wissenschaftliche Plattform für morphometrische Analysen.

Serie: 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.

Eine isolierte Retinal Vorbereitung auf leichte Ansprache aus genetisch markierten retinalen Ganglienzellen Rekord

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

Forschung

Neurowissenschaften

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.

Protokoll zur dreidimensionalen konfokalen morphometrische Analyse der Astrozyten

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.

Immunostaining von Biocytin gefüllte und verarbeitete Abschnitte für Marker Neurochemisches

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.

Microstate und Omega Komplexität Analysen der ruhenden Zustand Elektroenzephalographie

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.

Vorbereitung von Maus-Retinal-Cryo-Sektionen für die Immunhistochemie

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.

Vorbereitung des Neugeborenen Ratsprofels für die ultrastrukturelle Morphometrische Analyse der Verbreitung synaptischer Bläschen an Nerven-Terminals

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.

Sammlung von gefrorenen Nagetier-Hirnregionen für Downstream-Analysen

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.

Vorbereitung der Stimmlippe der Ratte für neuromuskuläre Analysen

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.

Dissektion einzelner Skelettmuskelfasern für immunfluoreszierende und morphometrische Analysen von neuromuskulären Verbindungen am ganzen Berg

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.