Bu laboratuvarda göz araştırma çalışmaları Amerikan Sağlık Yardımcısı Vakfı, Açelya (1972) Bağış Fonu ve HKU Küçük Projeler Fonu (20097176185) tarafından desteklenmektedir.

1. Araçlar Mikroskop, Nikon Stereo Araştırmacı, MBF Bioscience - MicroBrightField, Inc 2. Hazırlık <ol><li> Herhangi bir morfometrik analiz çalışmaya başlamadan önce, her bir retina örneği 4 mikron kalınlığında kesitler ve H &amp; E boyama uğrar. </li><li> Üst periferal bölgesi, üst merkezi bölge, düşük periferal bölgeye ve alt merkezi bölge - retina bölümünde 4 bölgeye ayrılmaktadır. </li><li> Bölgelerde tanımladıktan sonra, her bölgenin bir görüntü Nikon parlak ışını mikroskop altında 10x40 büyütülerek elde edilir. </li><li> Şimdi, retinanın morfometri analiz etmek için hazırız. </li></ol> 3. INL kalınlığı ölçümü <ol><li> Her bir bölge içinde INL kalınlığının dört ölçümün ölçüldü. Dört satır INL dış sınırına kontur başlangıca dik çizilecektir. </li><li>Stereo Investigator adlı yazılımı açın ve Ana Araç Çubuğu ile 40x Lens seçin. </li><li> Sekmesinde &quot;File&quot; altında &quot;Resim Aç&quot; seçeneğini seçin. Analiz etmek istediğiniz bir arzu retina bölgenin bir görüntü seçin. </li><li> Ana Araç Çubuğu Açılan liste kutusundan kontur çizgisi türünü seçin ve &quot;Auto Move&quot; düğmesine basın. </li><li> Sağ izleme penceresinde tıklatın ve &quot;Basit tıklayın İzleme&quot; seçeneğini seçin. </li><li> INL iç sınırı boyunca kontur çizgi çizin. </li><li> Çizim yapılır, sağ izleme penceresinde tıklatın ve &quot;Open End Kontur&quot; seçeneğini seçin. </li><li> Işaretleyici Araç çubuğunda bir işaretleyici seçin ve kavşak noktalarında işaretleri ekleyebilirsiniz. </li><li> Tüm işaretleri ekledikten sonra, Marker Araç Çubuğu de işaretleyici simgesi kaldırın. </li><li> Belirteçlerin birinin merkezinde bir imleç yerleştirin. </li></ol><img src="/files/ftp_upload/3377/3377fig1.jpg" alt="Şekil 1" /><ol start="11"><li> &quot;Hızlı SeçAraçlar &quot;&quot; sekmesi altında &quot;Açı ölçün. </li></ol><img src="/files/ftp_upload/3377/3377fig2.jpg" alt="Şekil 2" /><ol start="12"><li> Sağ izleme penceresinde tıklayın ve &quot;Sürekli&quot; seçeneğini kaldırın. </li><li> Birinci çizgi parçasının bir uç için seçilen işaretleyici bitişik olan bir işaretleyici, merkezinde tıklaması ile bir açı ölçün. </li></ol><img src="/files/ftp_upload/3377/3377fig3.jpg" alt="Şekil 3" /><ol start="14"><li> Ardından, ilgili belirteç merkezine fareyi tekrar tıklayın. </li><li> INL dış sınırına lastik bant hattı uzatın. </li><li> 90 ° ulaşılana kadar açısını ayarlayın. </li></ol><img src="/files/ftp_upload/3377/3377fig4.jpg" alt="Şekil 4" /><ol start="17"><li> Istenilen konumda sol tıklayın. Bir açı ölçümü gösteren bir iletişim kutusu tıklayın hemen sonra açılacaktır. </li><li> BasınYine fare tutarak &quot;Enter&quot; tuşuna. </li></ol><img src="/files/ftp_upload/3377/3377fig5.jpg" alt="Şekil 5," /><ol start="19"><li> Bir çizgi bir ucundan itibaren yeni seçilen sonuna kadar çekilir, böylece izleme penceresinin tıklayın bırakılır. </li><li> Sağ izleme penceresinde tıklayın ve &quot;Open End Kontur&quot; seçeneğini seçin. </li></ol><img src="/files/ftp_upload/3377/3377fig6.jpg" alt="Şekil 6" /><ol start="21"><li> Daha önce tarif edildiği gibi aynı şekilde farklı pozisyonlarda diğer çizgi çizer. </li><li> Bütün çizgiler çizdikten sonra, düzenleme modunu seçin ve buna göre 4 çizgi parçaları yeniden adlandırın. </li><li> Dört ölçümleri &quot;Kontur Ölçme&quot; butonuna basarak elde edilebilir. </li><li> Bu 4 ölçüm seçin ve &quot;Panoya Kopyala&quot; tıklayarak bir tabloya yapıştırabilirsiniz. </li><li> Sekmesini seçin &quot;Dosya&quot; nın altında &quot;Farklı Kaydet&quot;. Izleme verileri bir veri dosyası olarak kaydedilebilir. </li></ol><ol><li> Sekmesinde &quot;File&quot; altında &quot;Resim Aç&quot; seçeneğini seçin. Analiz etmek istediğiniz bir arzu retina bölgenin bir görüntü seçin. </li><li> Ana Araç Çubuğu Açılan liste kutusundan kontur çizgisi türünü seçin. </li><li> Sinir lifi tabakası (NFL) sınır boyunca bir kontur çizgi çizin. </li><li> Seçeneği &quot;End Open Çevresi&quot; ile izleme End. </li><li> Her sonunda bir hattın ucunda marker ucuna yakın konumda başka bir ekleyin. </li><li> Işaretleyici Araç çubuğunda işaretleyici simgesi kaldırın. </li><li> Ucunda belirteçlerin merkezinde bir imleci yerleştirin. </li><li> Sekmesinde &quot;Araçlar&quot; altında &quot;Hızlı Measure Angle&quot; seçeneğini seçin. </li><li> Sağ izleme penceresinde tıklayın ve &quot;Sürekli&quot; seçeneğini kaldırın. </li><li> Lastik bant hatları ayarlayın ve 90 ° lik bir açı ölçer. </li><li> Bir iletişim kutusu kadar uzattı sonra &quot;Enter&quot; tuşuna basın. </li><li> Sol tıklama tyeni bir satır dik ucundan uzatılabilir böylece o pencere izleme. </li><li> Bir başka ucunda aynı şekilde yeni bir çizgi çizin. </li><li> İki sınırları RGCs sayısı sayılır, içinde oluşturulur. </li><li> Hücre dokunuşlar sayılmaz bir dışlama çizgisi olarak bir sınırı seçin. </li><li> Işaretleyici bir türünü seçin ve her canlı hücre bir takın. </li><li> Ölü hücreleri için bir belirteç başka bir türü seçin. </li><li> Yerleştirilen işaretleri her tür sayısı işaretleyici Araç çubuğunda buna göre işaretleyici simgesi yanında görüntülenir. </li><li> Düzenleme modunu seçin ve kontur temel almak. Sağ izleme penceresinde tıklatın ve hat yeniden adlandırın. </li><li> Kontur taban uzunluğu &quot;Kontur Ölçme&quot; butonuna basarak elde edilebilir. </li><li> Hücrelerin sayısının daha sonra uzunluğunun mm başına ifade edilebilir. </li><li> Sekmesini seçin &quot;Dosya&quot; nın altında &quot;Farklı Kaydet&quot;.izleme ve marker veriler bir veri dosyası olarak kaydedilebilir. </li></ol> 5.. Bir RGC hücre hücre boyutu Ölçme <ol><li> Sekmesinde &quot;File&quot; altında &quot;Resim Aç&quot; seçeneğini seçin. Analiz etmek istediğiniz bir arzu retina bölgenin bir görüntü seçin. </li><li> Canlı bir hücreye Zoom ve Ana Araç Çubuğu Açılan liste kutusundan kontur çizgisi türünü seçin. </li><li> Hücrenin çevresi boyunca bir kontur çizgi çizmek. </li><li> Sonunda ilgili olarak, sağ izleme penceresini tıklatın ve &quot;End yakın kontur&quot; seçeneğini seçin. </li><li> Hücre alanı Kontur Ölçüm iletişim kutusundaki Alan kalemi altında gösterilir. </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>

Yazarlar ifşa hiçbir şey yok.

1. Daha doğru bir kalınlığı ölçümü nasıl elde edilir? Bu butonu &quot;Zoom&quot; e tıklayarak görüntüyü büyütmek ve izleme penceresinde sol tıklama yapabilirsiniz. INL sınır açıkça görülebilir. Çizginin ucu INL dış sınırında değilse, biz Düzenleme modu altında çizgi ayarlayabilirsiniz. Ilgili satırı alıp sonra sağ izleme penceresi tıklayın ve &quot;Seçilmiş Kontur Nokta Ekle&quot;. Eklenen noktası aynı hat boyunca olmalıdır sırada bir nokta dış ...

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> Reaktif Adı </td><td> Tip </td><td> Şirket </td><td> Katalog numarası </td></tr><tr><td> Stereo Investigator </td><td> MBF Bioscience Analiz Yazılımı </td><td> MicroBright Alan </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

Retinal bölümlerin morfo analizleri retinal hastalıklar inceleyerek kullanılmıştır. Örnekler için, nöronal hücrelerde önemli ölçüde N-metil-D-aspartat ile sıçan modelinde retina ganglion hücre tabakası (RGCL) kayboldu (NMDA) eksitotoksisite 1, retinal iskemi-reperfüzyon yaralanması 2 ve glokom 3-indüklenen. INL ve iç pleksiform tabakası azaltılması (IPL) kainik asit-aracılı glutamat eksitotoksi 4 maruz sıçan gözünde sitikolin tedavisi ile tersine döndü kalınlıkta. RGC yoğunluğu ve soma boyutlarda Alterasyon göziçi basınç 3,5,6 gözlerde farklı ilaç tedavileri ile gözlenmiştir. Bu nedenle, retina morphometries analiz objektif yöntemlerle sahip retinal patolojiler ve tedavi stratejilerinin etkinliğini değerlendirmek büyük önem olabilir. Retina yapısı çoklu katmanlar ve nöronlar exi birkaç farklı olduğunuretinada st. Gibi farklı katmanlardan hücre sayısı, hücre boyutu ve kalınlığı gibi retina morfometrik parametreler hücre kültür sistemi daha karmaşıktır. Erken, bu parametrelerin diğer ticari görüntüleme yazılımı kullanılarak tespit edilebilir. Değerleri göreceli değerinin normalde, ve kesin bir değere değişen daha doğru hesaplama gerekebilir. Ayrıca, hücre boyutu ve morfolojisi izleme, özellikle kronik glokom modeli, doğru ve istatistik analiz için yeterince hassas olmayabilir. Bu protokolde kullanılan ölçümler daha hassas ve kolay bir şekilde sağlanmaktadır. Ve hücre çizgisi ve büyüklüğü mutlak uzunluğu, doğrudan veya diğer dosyaları kopyalanacak kolay kaydedilmiştir. Örneğin, INL yılında iç ve dış en çekirdeklerin marjı takip ve kalınlığını ölçmek için 90 derecelik bir açı çizmek için yazılım kullanarak bir çizgi oluşturdu. Yazılım yardımı olmadan, belki eğik çizgi ve retina kalınlığı değişen olabilir ikenBireysel gözlemciler arasında tekrarlanabilir olmayabilir. Buna ek olarak, RGCs sayısını ve ayrıca yoğunluğu belirlenebilir. Bu protokol başarıyla, retinanın özellikleri miktarlarının değişkenliği azalır minimal değişiklikler tespit hassasiyetini artırır. Bu video retina bölümleri morfometrik analizler üç tip gösterecektir. Bunlar, INL kalınlığının ölçülmesi RGCs sayısı miktarının ve mutlak değerinin RGCs boyutları ölçüm içerir. Bu üç analizleri Stereo Araştırıcı (- MicroBrightField, Inc MBF Bioscience) ile gerçekleştirilir. Tekniği morfometrik analizler için basit ama bilimsel bir platform sunabilir.

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

Morphometric Analyses of Retinal Sections

Bu video retina ganglion hücrelerinin sayısı (RGCs) ve niceleme RGCs boyutları ölçülerek, iç nükleer tabaka kalınlığı ölçümü de retinanın morfometrik analizler, üç tip gösterir. Tekniği morfometrik analizler için basit ama bilimsel bir platform sunabilir.

Retina Bölüm morfometrik Analizleri

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 Görüntüleme.  The University of Hong Kong. Bu video retina ganglion hücrelerinin sayısı (RGCs) ve niceleme RGCs boyutları ölçülerek, iç nükleer tabaka kalınlığı ölçümü de retinanın morfometrik analizler, üç tip gösterir. Tekniği morfometrik analizler için basit ama bilimsel bir platform sunabilir.

Video: Morphometric Analyses of Retinal Sections

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

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

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

Genetik Etiketli Retina Ganglion Hücreleri Işık Tepkisi Record İzole Retina Hazırlık

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

Nörobilim

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.

Astrositler Üç boyutlu Konfokal Morfometrik Analizi Protokolü

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.

Nörokimyasal markerleri Biocytin dolu ve İşlenmiş Bölümler immün

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 ve dinlenme-devlet elektroansefalografi Omega karmaşıklık analizleri

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.

Immünhistokimya için fare retinal Cryo-bölümler hazırlanması

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.

Sinir terminallerinde sinaptik vezikül dağıtımının Ultrastrik morfometrik analizi için yenidoğan Rat beyin dokusunun hazırlanması

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.

Downstream Analizleri için Dondurulmuş Kemirgen Beyin Bölgeleri Koleksiyonu

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.

Nöromüsküler Analizler için Sıçan Vokal Kıvrımının Hazırlanması

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.

Tüm Mount Nöromüsküler Kavşakların İmmünofluoresan ve Morfometrik Analizleri için Tek İskelet Kas Liflerinin Diseksiyonu

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.