この研究室で目の研究の仕事はアメリカの健康アシスタント財団、ツツジ（1972）基本財産基金、およびHKU小規模プロジェクト基金（20097176185）でサポートされています。

1。ツール顕微鏡、ニコンステレオ調査官、MBFバイオサイエンス - MicroBrightField株式会社 2。準備<ol><li>任意の形態学的分析の作業を行う前に、それぞれの網膜サンプルは、4ミクロンの厚さで切片であり、H＆E染色を受ける。 </li><li>上部の周辺領域、上部の中央部、下側の周辺領域と、下部中央の領域 - 網膜セクションは4つの領域に分割されています。 </li><li>領域を定義した後、各領域の画像は、ニコンの明るい光顕微鏡下で10X40倍率で取得されます。 </li><li>今、私たちは、網膜の形態計測分析を行うことができます。 </li></ol> 3。 INLの厚さを測定<ol><li>各地域のINLの厚さの4つの測定値を測定した。 4行は、INLの外側の境界に輪郭のベースラインから垂直に描画されます。 </li><li>ステレオ捜査官と呼ばれるソフトウェアを開き、メインツールバーから40倍レンズを選択します。 </li><li>タブの &quot;ファイル&quot;の下の &quot;画像を開く&quot;を選択します。あなたが分析したいことが望ま網膜領域の画像を選択します。 </li><li>メインツールバーのドロップダウンリストボックスから、輪郭線の種類を選択し、 &quot;自動移動&quot;ボタンを押してください。 </li><li>右のトレースウィンドウ内をクリックし、 &quot;単純なクリックしてトレース&quot;を選択してください。 </li><li> INLの内側の境界に沿って輪郭線を描画します。 </li><li>描画が完了したら、右側のトレースウィンドウ内をクリックし、 &quot;終了オープン輪郭&quot;を選択します。 </li><li>マーカーツールバーでマーカーを選択して、接合点にマーカーを追加します。 </li><li>すべてのマーカーを追加した後、マーカーツールバーでマーカーのアイコンを選択解除します。 </li><li>マーカーの1つの中心にカーソルを置きます。 </li></ol><img src="/files/ftp_upload/3377/3377fig1.jpg" alt="図1" /><ol start="11"><li> &quot;クイックを選択します。ツール &quot;&quot;タブの下で、 &quot;角度を測定します。 </li></ol><img src="/files/ftp_upload/3377/3377fig2.jpg" alt="図2" /><ol start="12"><li>右のトレースウィンドウ内をクリックし、 &quot;連続&quot;オプションの選択を解除します。 </li><li>最初の行セグメントの終点のために選択マーカーに隣接しているマーカーの中心にクリックして、角度を測定します。 </li></ol><img src="/files/ftp_upload/3377/3377fig3.jpg" alt="図3" /><ol start="14"><li>その後、当該マーカーの中央にマウスを移動し、再度クリックします。 </li><li> INLの外側の境界線にゴムバンド線を拡張します。 </li><li> 90°に達するまでの角度を調整します。 </li></ol><img src="/files/ftp_upload/3377/3377fig4.jpg" alt="図4" /><ol start="17"><li>任意の位置で左クリックします。角度測定を示すダイアログボックスが右クリックした後にポップアップ表示されます。 </li><li>押すまだマウスを押しながら &quot;Enter&quot;キーを。 </li></ol><img src="/files/ftp_upload/3377/3377fig5.jpg" alt="図5" /><ol start="19"><li>ラインが​​1つの端から新たに選択最後に描画されるように、トレースウィンドウを左クリックしてください。 </li><li>右のトレースウィンドウ内をクリックし、 &quot;終了オープン輪郭&quot;を選択します。 </li></ol><img src="/files/ftp_upload/3377/3377fig6.jpg" alt="図6" /><ol start="21"><li>前述のようにまったく同じ方法で異なる位置で他の線を描画します。 </li><li>すべての行を描画した後、編集モードを選択し、それに応じて4ラインセグメントの名前を変更します。 </li><li> 4つの測定は、 &quot;輪郭測定&quot;ボタンを押すことによって得ることができます。 </li><li>これらの4つの測定値を選択し、 &quot;クリップボードにコピー&quot;をクリックしてスプレッドシートに貼り付けることができます。 </li><li>選択したタブの &quot;ファイル&quot;の下に &quot;名前を付けて保存&quot;。トレースデータは、データファイルとして保存することができます。 </li></ol><ol><li>タブの &quot;ファイル&quot;の下の &quot;画像を開く&quot;を選択します。あなたが分析したいことが望ま網膜領域の画像を選択します。 </li><li>メインツールバーのドロップダウンリストボックスから、輪郭線の種類を選択します。 </li><li>神経線維層（NFL）の境界に沿って輪郭線を描画します。 </li><li>オプションの &quot;Endオープン輪郭&quot;でトレースを終了します。 </li><li>それぞれの終わりに、1行の先端にマーカーと先端に近い位置で別のを追加します。 </li><li>マーカーツールバーのマーカーアイコンを選択解除します。 </li><li>先端のマーカーの中心にカーソルを置きます。 </li><li>タブの &quot;ツール&quot;の下にある &quot;クイック測定角度&quot;を選択します。 </li><li>右のトレースウィンドウ内をクリックし、 &quot;連続&quot;オプションの選択を解除します。 </li><li>ゴムバンド線を調整し、90°の角度を測定します。 </li><li>ダイアログボックスがポップアップされた後、 &quot;Enter&quot;キーを押します。 </li><li>左クリックトン彼は、新し​​い行が垂直に先端から延長することができるようにウィンドウをトレースします。 </li><li>もう終わりにまったく同じ方法で別の新しい線を引く。 </li><li> 2つの境界は、RGCの数がカウントされている内に作成されます。 </li><li>セルタッチがカウントされません除外ラインとして1つの境界線を選択してください。 </li><li>マーカーのいずれかのタイプを選択し、各ライブセルに1を添付してください。 </li><li>死んだ細胞のマーカーの別のタイプを選択します。 </li><li>に設定されたマーカーの種類ごとの数は、マーカーのツールバーに応じてマーカーアイコンの横に表示されます。 </li><li>編集モードを選択し、輪郭のベースラインを拾う。右のトレースウィンドウ内をクリックし、行の名前を変更します。 </li><li>コンターラインの長さは、 &quot;輪郭測定&quot;ボタンを押すことによって得ることができます。 </li><li>細胞の数は、長さのmm当たり表現することができます。 </li><li>選択したタブの &quot;ファイル&quot;の下に &quot;名前を付けて保存&quot;。ザトレースとマーカデータは、データファイルとして保存することができます。 </li></ol> 5。 RGCセルのセルサイズを測定する<ol><li>タブの &quot;ファイル&quot;の下の &quot;画像を開く&quot;を選択します。あなたが分析したいことが望ま網膜領域の画像を選択します。 </li><li>生細胞でのズームとメインツールバーのドロップダウンリストボックスから、輪郭線の種類を選択します。 </li><li>セルの円周に沿って輪郭線を描画します。 </li><li>約終了時には、右のトレースウィンドウをクリックすると &quot;Endに近い輪郭&quot;を選択してください。 </li><li>セルの面積は輪郭測定]ダイアログボックスでエリアの項目の下に表示されます。 </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>

著者らは、開示することは何もありません。

1。より正確な厚さの測定値を得る方法は？ あなたは、トレースウィンドウ内のボタンと左クリック &quot;ズーム&quot;をクリックして画像を拡大することができます。 INL境界線がはっきりと見ることができます。ラインの先端がINLの外側の境界でない場合は、我々は、編集モードで行を調整することができます。関係線を選んだ後、右のトレースウィンドウをクリックして ...

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> 試薬の名前 </td><td> タイプ </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td>ステレオ捜査官</td><td> MBFバイオサイエンス解析ソフトウェア</td><td> MicroBrightフィールド</td><td></td></tr><tr><td>顕微鏡</td><td>オリンパスBX51 </td><td>オリンパス株式会社</td><td> BX51 </td></tr></tbody></table>

morphometric analyses of retinal sections

網膜切片の形態学的分析は、網膜疾患を調べるに使用されている。例については、神経細胞が大幅にN-メチル-D-アスパラギン酸（NMDA）誘発性興奮1、網膜虚血再灌流障害2、緑内障3ラットモデルの網膜神経節細胞層（RGCL）で失われた。 INLおよび内網状層の減少（IPL）は、カイニン酸介在型グルタミン酸興奮毒性4に受けたラットの目にシチコリン治療と逆転した厚さ。 RGC密度とソーマのサイズの変化は、眼圧上昇3,5,6の目に別の薬物治療で観察した。したがって、網膜morphometriesを分析する客観的方法を持つことは、網膜の病態と治療戦略の有効性を評価する上で大きな意義であるかもしれない。 網膜の構造は、マルチレイヤーとニューロンEXIいくつかの異なる種類です。網膜の聖。そのような細胞数、細胞の大きさと異なった層の厚さとして、網膜の形態学的パラメータは、細胞培養系よりも複雑です。早い段階で、これらのパラメータは他の市販のイメージングソフトウェアを使用して検出することができます。値は相対値で、通常であり、正確な値に変更すると、さらに正確な計算が必要な場合があります。また、セルサイズと形態のトレースは、特に慢性緑内障モデルでは、正確な統計分析のための十分な感度ではありません。このプロトコルで使用されている測定値は、より正確かつ簡単な方法を提供しました。セルの行とサイズの絶対的な長さは、直接他のファイルにコピーすることが簡単に報告することができます。たとえば、我々は、INLの内側と外側の最も核のマージンを追跡して、厚さを測定するために90度の角度を描画するソフトウェアを使用してラインを形成した。ソフトウェアの助けを借りずに、多分斜線と網膜の厚さの変化がしれませんが個々のオブザーバーの間で再現性がありません。さらに、RGCの数と密度も定量することができる。このプロトコルは、成功すると、網膜の機能を定量の変動を減少させる最小限の変更を検出する感度を向上させます。 このビデオでは、網膜切片の形態学的分析の3種類のデモンストレーションを行います。彼らは、RGCの数を定量化し、絶対値でRGCのサイズを測定し、INLの厚さを測定しています。これらの3つの解析は、ステレオ調査官（ - MicroBrightField株式会社MBFバイオサイエンス）を用いて行われています。技術は、形態学的解析のためのシンプルでありながら科学的なプラットフォームを提供することができます。

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

Morphometric Analyses of Retinal Sections

このビデオでは、内顆粒層の厚さ、網膜神経節細胞（RGC）の数を定量化し、RGCのサイズを測定する測定含まれる網膜の形態学的分析、3つのタイプを示しています。技術は、形態学的解析のためのシンプルでありながら科学的なプラットフォームを提供することができます。

網膜切片の形態学的解析

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 ビュー.  The University of Hong Kong. このビデオでは、内顆粒層の厚さ、網膜神経節細胞（RGC）の数を定量化し、RGCのサイズを測定する測定含まれる網膜の形態学的分析、3つのタイプを示しています。技術は、形態学的解析のためのシンプルでありながら科学的なプラットフォームを提供することができます。

ビデオ: 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.

遺伝的に標識された網膜神経節細胞からの光は応答を記録するために隔離された網膜の準備

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

神経科学

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.

アストロサイトの三次元共焦点形態学的分析のためのプロトコル

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.

神経化学的マーカーのためのビオシチンで満たされたと加工セクションの免疫染色

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.

微小状態と静止状態の脳波のオメガ複雑さ解析

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.

免疫組織化学用マウスレチンクライオ切片の調製

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.

Ultrastructural のための新生ラット脳組織の調製 Morphometric 神経末端におけるシナプス小胞分布の解析

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.

下流解析のための凍結げっ歯類の脳領域の収集

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.

神経筋解析のためのラット声帯の調製

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.

全山神経筋接合部の免疫蛍光解析と形態測定解析のための単一骨格筋線維の解剖

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.