この作品は中国の国家自然科学基金 (許可番号 31400936、31460260) によって支えられた、自然科学財団の江西省の中国 (20171BAB215020)。我々 はまた、この作業を支援する南昌大学とクイーン クイーンメアリー、ロンドン大学との共同プログラムを感謝します。

ここで説明したすべてのメソッドは、動物のケアと使用南昌大学委員会によって承認されています。
1. 機器をアセンブルし、ミクロトームを接続
<ol><li>要件 (補助図 1) あたりパラメーターを設計します。</li>
	<li>アクリル板を製造するローカル工場にパラメーターを送信します。</li>
	<li>シーケンスのすべての部品を組み立てる: クロロホルムを使用してタンクに 7 商業アクリル板を組み合わせてセクション チャネルと水お風呂 (図 1)。 注意: 光と酸素を満たしているとき、クロロホルムは有毒物質を生成します。ヒューム フードの装置を組み立てます。</li>
	<li>アクリル板 #1 (図 2 a,矢印) に穴を介して管状電気加熱要素をインストールします。</li>
	<li>温度コント ローラーをインストールするのには、電気ドリルを使用して #2 アクリル板に小さな穴を作成し、温度検出器 (図 2 a,矢印) をインストールします。タンク (図 2 a) にデジタル サーモスタットをインストールします。 注: 温度検出器は水面下 1 cm 以内にする必要があります。</li>
	<li>電源アクセスを提供するために、機器の電源を入れる前に 24 V 以下の電圧を調整します。</li>
	<li>ミクロトームから切片屑トレーを取り外します。</li>
	<li>中立的なシリコーン ・ シーラント (図 2 b) ミクロトーム ナイフ ホルダーを含むセクション チャネルにリンクします。 注: 刃の上の上の中立シリコーン ・ シーラント中傷されず古い刃は簡単に交換できるようにします。</li>
	<li>クロロホルムと固化する中立的なシリコン シール剤の時間を許可するために一晩乾燥させます。</li>
	<li>ブレード ホルダーに刃をインストールします。(図 2) 風呂の水に水を注ぐ。水表面だけブレード (図 2 D,アロー ・ ヘッド) の上部に触れる必要があります。</li>
</ol>2. パラフィン切片
注: 破片は、多くの場合ワックスのブロックと水の表面の上下端に蓄積します。この残骸は、定期的にクリアする必要があります。
<ol><li>
従来のパラフィン切片10を実行します。
	<ol>
<li>回転式ミクロトームのパラフィン包埋標本をクランプし、ブレード ホルダーにブレードを配置します。</li>
		<li>ハンド ホイールを切り、標本をセクションします。</li>
		<li>パラフィン切片のリボンを 38.5 ° C の温水浴に転送します。</li>
		<li>顕微鏡にセクションを展開した後の水面からスライドをピックアップします。</li>
		<li>42 ° C のオーブンでスライドを一晩乾かします。</li>
	</ol>
</li>
	<li>
改良されたパラフィン切片 (図 3)
	<ol>
<li>機器の電源を。</li>
		<li>温度検出スイッチでヒーターを開き、40.0 ° C 1 時間事前に水を暖めるために 38.0 ° C 間ターゲットの温度を設定します。</li>
		<li>パラフィン包埋にしたパラフィン ブロックを変更します。ミクロトームのカセット クランプにパラフィン ブロックを配置し、それをロックします。 注意: パラフィン ブロックの優れたと劣るボーダーは、円弧を形成し、狭いセクション チャネルを通過することができないからセクション リボンを防ぐために水平方向に変更ください。</li>
		<li>切断より厚いセクションでサンプルの迅速なアプローチします。</li>
		<li>適切なセクションの厚さ (成体マウスの脳、成体マウス腎臓、胎児マウスの脳に 10 μ m、ゼブラフィッシュの目 4 μ m) を調整します。ハンド ホイールを切り、セクションは、セクション チャネルと風呂に自動的に入力されます。</li>
		<li>ゆっくりと均一な切断ストローク (図 3 a) で標本をセクションします。 注: ハンド ホイールが数分間停止した後の最初のセクションが変更されます。セクションは連続した傷がある場合、替刃を置き換える必要があります。</li>
		<li>ライン (図 3 b、3 C) で浮かぶ風呂室にセクションをガイドします。 注: 場合は head セクションは、チャンバー壁に準拠して、浮いた状態を保つにブラシ ガイドを使用します。</li>
		<li>顕微鏡スライド上にセクションを遵守します。セクションの後完全に風呂の水、ピンセットで別のいくつかのセクションで展開します。顕微鏡のスライド (図 3 D) に風呂の水からそれらをピックアップします。 注: セクションする必要がありますピックアップする水のバスの後方からセクション チャネルと水お風呂の前のそれらがまだできないので完全に展開。</li>
		<li>一晩 42 ° C のオーブンでそれらを配置することによって、スライドを乾燥します。スライドは、無期限に室温で格納されることができます。</li>
	</ol>
</li>
	<li>ヘマトキシリン ・ エオジン (HE) 染色法の改良8の評価のために使用します。</li>
</ol>

<ol>
	<li>Chung, K., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CLARITY+for+mapping+the+nervous+system.">CLARITY for mapping the nervous system.</a> Nature Methods. 10 (6), 508-513 (2013).</li><li>Fujita, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+neuron+differentiation+in+the+central+nervous+system+by+tritiated+thymidine+autoradiography.">Analysis of neuron differentiation in the central nervous system by tritiated thymidine autoradiography.</a> Journal of Comparative Neurology. 122 (3), 311-327 (1964).</li><li>Mironov, V., Boland, T., Trusk, T., Forgacs, G., Markwald, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ+printing:+Computer-aided+jet-based+3D+tissue+engineering.">Organ printing: Computer-aided jet-based 3D tissue engineering.</a> Trends in Biotechnology. 21 (4), 157-161 (2003).</li><li>Fischer, A. H., Jacobson, K. A., Rose, J., Zeller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryosectioning+tissues.">Cryosectioning tissues.</a> Cold Spring Harbor Protocols. 3 (8), (2008).</li><li>Viebahn, C., Luttenberg, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+anti-roll+plate+as+a+remedy+for+the+ill-effects+of+electrical+charge+during+cryosectioning.">A modified anti-roll plate as a remedy for the ill-effects of electrical charge during cryosectioning.</a> Journal of Histochemistry and Cytochemistry. 37 (7), 1157-1160 (1989).</li><li>Onozato, M. L., Hammond, S., Merren, M., Yagi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+a+completely+automated+tissue-sectioning+machine+for+paraffin+blocks.">Evaluation of a completely automated tissue-sectioning machine for paraffin blocks.</a> Journal of Clinical Pathology. 66 (2), 151-154 (2013).</li><li>Sabaliauskas, N. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+zebrafish+histology.">High-throughput zebrafish histology.</a> Methods. 39 (3), 246-254 (2006).</li><li>Chen, T. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hybrid-Cut:+An+Improved+Sectioning+Method+for+Recalcitrant+Plant+Tissue+Samples.">Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples.</a> Journal of Visualized Experiments. (117), e54754 (2016).</li><li>Kucherenko, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paraffin-Embedded+and+Frozen+Sections+of+Drosophila+Adult+Muscles.">Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles.</a> Journal of Visualized Experiments. (46), e2438 (2010).</li><li>Cornell, W. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paraffin+Embedding+and+Thin+Sectioning+of+Microbial+Colony+Biofilms+for+Microscopic+Analysis.">Paraffin Embedding and Thin Sectioning of Microbial Colony Biofilms for Microscopic Analysis.</a> Journal of Visualized Experiments. (133), e57196 (2018).</li><li>Whiteland, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemical+detection+of+T-cell+subsets+and+other+leukocytes+in+paraffin-embedded+rat+and+mouse+tissues+with+monoclonal+antibodies.">Immunohistochemical detection of T-cell subsets and other leukocytes in paraffin-embedded rat and mouse tissues with monoclonal antibodies.</a> Journal of Histochemistry and Cytochemistry. 43 (3), 313-320 (1995).</li><li>Tucker, D. K., Foley, J. F., Bouknight, S. A., Fenton, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sectioning+Mammary+Gland+Whole+Mounts+for+Lesion+Identification.">Sectioning Mammary Gland Whole Mounts for Lesion Identification.</a> Journal of Visualized Experiments. (125), e55796 (2017).</li><li>Venegas-Pino, D. E., Banko, N., Khan, M. I., Shi, Y., Werstuck, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Analysis+and+Characterization+of+Atherosclerotic+Lesions+in+the+Murine+Aortic+Sinus.">Quantitative Analysis and Characterization of Atherosclerotic Lesions in the Murine Aortic Sinus.</a> Journal of Visualized Experiments. (82), e50933 (2013).</li><li>Lau, S. K., Chu, P. G., Weiss, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD163:+A+specific+marker+of+macrophages+in+paraffin-embedded+tissue+samples.">CD163: A specific marker of macrophages in paraffin-embedded tissue samples.</a> American Journal of Clinical Pathology. 122 (5), 794-801 (2004).</li><li>Campbell-Thompson, M. L., Heiple, T., Montgomery, E., Zhang, L., Schneider, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staining+Protocols+for+Human+Pancreatic+Islets.">Staining Protocols for Human Pancreatic Islets.</a> Journal of Visualized Experiments. (63), e4068 (2012).</li></ol>

著者は、このデバイスの特許を持っているし、競争の興味を宣言しません。

パラフィン セクション形態を改善し、従来のパラフィン切片中の無駄な時間の問題を解決するには、改良されたパラフィン切片作製法切削と展開を組み合わせたを作成しました。この改良法は、セクション チャネル、水浴と温度検出スイッチとヒーターで構成されるシンプルな機器に依存します。セクション リボン セクション チャネルを介して水の風呂に入るし、自動的に切削しながら?...

形態学的研究は、生物学の研究が重要です。新技術は、全体の組織または生物1,2,3、薄い部分に試料を切断から直接彼らの目標は続いて染色を観察する研究者を許可しているが、プライマリのまま法者は唯一組織形態も組織に直接ターゲット蛋白質。光学顕微鏡を使用して、3 つのセクション タイプ: パラフィン、冷凍、およびグリコールメタクリル。セクショニングは共通組織抗原性を保護するため、試料の準備は簡単です、保有組織形態が悪いと薄い断面4,5には不向き。パラフィン切片は、保存状態の良い形態を展示するため最もよく使用される方法です。標本は完全に脱水し、ワックスに埋め込まれた、パラフィン ブロックに無期限に格納できます。また、パラフィン切片さらに実験の生物学的プローブ アクセスを改善し、Z 方向に細胞層オーバーレイを削減する薄いセクションを生成します。
しかし、従来のパラフィン切片がかかり、オペレーターのスキルを要求します。パラフィン切片を固定、脱水、埋め込み、切り取り、およびフローティング受けます。重要なは、風呂の水にナイフ ホルダーからセクション リボンを転送するは、ジュニアのオペレーターのためには必要なは難しいです。乾いた空気で特にセクション リボン静電気によるねじれが、暖かい水面に展開しにくい。セクションを改善するために品質、氷の水でそれらを浸すことまたはミクロトーム近く加湿器で湿度を上げるワックス ブロックを冷却のミクロトーム刃パス間公開される組織表面を潤し、6、7をお勧めします.ハイブリッド パラフィン包埋、セクショニング8、および商業部門転送システム支援9パラフィン切片を改善するための新しいメソッドが含まれます。これらのメソッドは、部分的にパラフィンを向上させる彼らははるかに厄介な断面を作る、商務部搬送システムは高価なセクショニング速度と品質。
このプロトコルでは、簡単、安価で柔軟な装置ステップバイ ステップ、回転式ミクロトームのナイフ ホルダーに接続することができますを作成する方法を示します。この装置は、温度検出スイッチ付きのヒーター、水風呂セクション チャネルで構成されます。切断した後、セクションの数十はセクション チャネルに流れるし、風呂の水、直接このように自動的に展開を入力します。これはパラフィン切片の効率が向上し、この技術をより便利になります。このメソッドよりアダルト マウス海馬セクション、成体マウス腎臓セクション、胚 15.5 日齢 (E15.5) マウスの脳のセクション、およびセクションを短い時間で収穫し、形態学的によりそのまま残った大人ゼブラフィッシュ眼を使用します。このメソッドは、加速されたパラフィン セクション区別の喪失を回避しながら断面を必要とする他の組織サンプルの使用もできます。

<table>
	<tbody>
		<tr>
			<td>Incubator</td>
			<td>Boekel Scientific</td>
			<td>133000-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol&#160;</td>
			<td>Sinopharm Chemical Reagent Co.,Lid</td>
			<td>64-17-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene&#160;</td>
			<td>Sinopharm Chemical Reagent Co.,Lid</td>
			<td>1330-20-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraplast</td>
			<td>Leica</td>
			<td>39601006</td>
			<td></td>
		</tr>
		<tr>
			<td>Heated Paraffin Embedding Module</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Commercial acrylic board</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trichloromethane</td>
			<td>Sinopharm Chemical Reagent Co.,Lid</td>
			<td>67-66-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Tubular electric heating element(12V 200W)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Temperature controller(12v 120w)</td>
			<td>Mingsuo</td>
			<td>XH-W3002</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotary microtome&#160;</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Neutral silicone sealant</td>
			<td></td>
			<td></td>
			<td>Link the water channel with the microtome knife holder</td>
		</tr>
		<tr>
			<td>Voltage transformer</td>
			<td>Dearll</td>
			<td>S-250-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable blade</td>
			<td>Accu-Edge</td>
			<td>4689</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin</td>
			<td>Baso Diagnostics Inc.</td>
			<td>BA-4025</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin&#160;</td>
			<td>Baso Diagnostics Inc.</td>
			<td>BA-4025</td>
			<td></td>
		</tr>
		<tr>
			<td>Microslide</td>
			<td>Sail Brand</td>
			<td>7105</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutral balsam</td>
			<td>Sinopharm Chemical Reagent Co.,Lid</td>
			<td>10004160</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslip&#160;</td>
			<td>Citoglas</td>
			<td>10212424C</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Xilong Chemical</td>
			<td>7647-01-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath for paraffin sections</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HistoCore Arcadia C&#160;- Cold Plate</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>paraffin repellent spray&#160;</td>
			<td>Thermo Scientific</td>
			<td>9990420</td>
			<td></td>
		</tr>
	</tbody>
</table>

the cutting and floating method for paraffin-embedded tissue for sectioning

組織学や病理学のパラフィン包埋組織の断面が使用されています。ただし、それは面倒です。この方法を改善するためにいくつかの民間企業は、液体の水を使用して複雑な部分転送システムを考案しています。この技術を簡素化し、我々 はカットとシンプルな恒温室; 内フローティングを組み合わせた自家製の機器を使用して単純なメソッドを作成したがって、セクションは自動的に水表面における水の風呂を入力します。アダルト ゼブラフィッシュ目マウス萌芽期の脳、成体マウス腎臓成体マウス脳海馬は、比較のため従来のパラフィン切片と提案手法の両方を使用して切られました。統計分析は、我々 の改良法時間を保存したし、高い品質のセクションを示しています。さらに、短時間で全体の標本のパラフィン切片ジュニア演算子の簡単です。

改善方法は、そのままパラフィン セクションの数を高めた。成体マウス海馬組織、成体マウス腎臓、マウス萌芽期の脳、ゼブラフィッシュの目のこの新しいメソッドをテストしました。タンクに水を追加し、40.0 ° c. に 38.0 ° C 水の温度に維持されました。準備した後、順次組織サンプル、切断され、従来区分と比較しています。新しいメソッドは、断面欠損を回避?...

Watch this Scientific Journal Video about The Cutting and Floating Method for Paraffin-embedded Tissue for Sectioning at JoVE.com

The Cutting and Floating Method for Paraffin-embedded Tissue for Sectioning

切削とフローティング区分のための組織のパラフィン包埋法

ここでは、パラフィン切片を改善するためのプロトコルを提案する.このメソッドは、切断と従来の方法に必要な転送処理を避けるために簡単な恒温室を使用して浮動小数点を兼ね備えています。その結果、そのままパラフィン切片数と効率が大幅に向上。

Sectioning of the paraffin-embedded tissue is widely used in histology and pathology. However, it is tedious. To improve this method, several commercial ...

the-cutting-floating-method-for-paraffin-embedded-tissue-for

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

18.2K ビュー.  Nanchang University. ここでは、パラフィン切片を改善するためのプロトコルを提案する.このメソッドは、切断と従来の方法に必要な転送処理を避けるために簡単な恒温室を使用して浮動小数点を兼ね備えています。その結果、そのままパラフィン切片数と効率が大幅に向上。

ビデオ: The Cutting and Floating Method for Paraffin-embedded Tissue for Sectioning

Vibratome Sectioning for Enhanced Preservation of the Cytoarchitecture of the Mammalian Organ of Corti

The mammalian organ of Corti is a highly ordered cellular mosaic of mechanosensory hair and nonsensory supporting cells (reviewed in 1,2).Visualization of this cellular mosaic often requires that the organ of Corti is cross-sectioned. In particular, the nonsensory pillar and Deiters' cells, whose nuclei are located basally with respect to the hair cells, cannot be visualized without cross-sectioning the organ of Corti. However, the delicate cytoarchitecture of the mammalian organ of Corti, including the fine cytoplasmic processes of the pillar and Deiters' cells, is difficult to preserve by routine histological procedures such as paraffin and cryo-sectioning, which are compatible with standard immunohistochemical staining techniques.
Here I describe a simple and robust procedure consisting of vibratome sectioning of the cochlea, immunohistochemical staining of these vibratome sections in whole mount, followed by confocal microscopy. This procedure has been used widely for immunhistochemical analysis of multiple organs, including the mouse limb bud, zebrafish gut, liver, pancreas, and heart (see 3-6 for selected examples). In addition, this procedure was sucessful for both imaging and quantitificaton of pillar cell number in mutant and control organs of Corti in both embryos and adult mice 7. This method, however, is currently not widely used to examine the mammalian organ of Corti. The potential for this procedure to both provide enhanced preservation of the fine cytoarchitecture of the adult organ of Corti and allow for quantification of various cell types is described.

A simple procedure of vibratome sectioning the organ of Corti, followed by immunohistochemistry and confocal microscopy is described. This procedure allows for improved preservation of the fine cytoarchitecture of the mammalian organ of Corti, and consequently allows for accurate quantification of cell types.

ビブラトームは、コルチ器の哺乳類器官の細胞構築の強化された保全のためセクショニング

The mammalian organ of Corti is a highly ordered cellular mosaic of mechanosensory hair and nonsensory supporting cells (reviewed in 1,2).Visualization of ...

神経科学

Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based multicolor labeling is challenging, especially when utilized for assessing interrelations between target proteins in the tissue with a high fat content such as the central nervous system (CNS).
Our protocol represents a refinement of the standard immunolabeling technique particularly adjusted for detection of both structural and soluble proteins in the rat CNS and peripheral lymph nodes (LN) affected by neuroinflammation. Nonetheless, with or without further modifications, our protocol could likely be used for detection of other related protein targets, even in other organs and species than here presented.

We here present an optimized, detailed protocol for double immunostaining in formalin-fixed, paraffin-embedded rat central nervous system (CNS) and peripheral lymph node (LN) tissue sections.

ラット中枢神経系および末梢リンパ節の組織切片での免疫組織化学的解析

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based ...

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have validated the utility of this method for enhancing neuronal preservation and overall brain slice viability. The implementation of this technique by early adopters has facilitated detailed investigations into brain function using diverse experimental applications and spanning a wide range of animal ages, brain regions, and cell types. Steps are outlined for carrying out the protective recovery brain slice technique using an optimized NMDG artificial cerebrospinal fluid (aCSF) media formulation and enhanced procedure to reliably obtain healthy brain slices for patch clamp electrophysiology. With this updated approach, a substantial improvement is observed in the speed and reliability of gigaohm seal formation during targeted patch clamp recording experiments while maintaining excellent neuronal preservation, thereby facilitating challenging experimental applications. Representative results are provided from multi-neuron patch clamp recording experiments to assay synaptic connectivity in neocortical brain slices prepared from young adult transgenic mice and mature adult human neurosurgical specimens. Furthermore, the optimized NMDG protective recovery method of brain slicing is compatible with both juvenile and adult animals, thus resolving a limitation of the original methodology. In summary, a single media formulation and brain slicing procedure can be implemented across various species and ages to achieve excellent viability and tissue preservation.

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol demonstrates the implementation of an optimized N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. A single media formulation is used to reliably obtain healthy brain slices from animals of any age and for diverse experimental applications.

最適化されたNを使用して急性脳スライスの準備-メチル-D-glucamine 保護回復法

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have ...

Utilizing 3D Printing Technology to Merge MRI with Histology: A Protocol for Brain Sectioning

Magnetic resonance imaging (MRI) allows for the delineation between normal and abnormal tissue on a macroscopic scale, sampling an entire tissue volume three-dimensionally. While MRI is an extremely sensitive tool for detecting tissue abnormalities, association of signal changes with an underlying pathological process is usually not straightforward. In the central nervous system, for example, inflammation, demyelination, axonal damage, gliosis, and neuronal death may all induce similar findings on MRI. As such, interpretation of MRI scans depends on the context, and radiological-histopathological correlation is therefore of the utmost importance. Unfortunately, traditional pathological sectioning of brain tissue is often imprecise and inconsistent, thus complicating the comparison between histology sections and MRI. This article presents novel methodology for accurately sectioning primate brain tissues and thus allowing precise matching between histology and MRI. The detailed protocol described in this article will assist investigators in applying this method, which relies on the creation of 3D printed brain slicers. Slightly modified, it can be easily implemented for brains of other species, including humans.

The overall goal of this protocol is to accurately align magnetic resonance imaging (MRI) image volumes with histology sections via the creation of customized 3D-printed brain holders and slicer boxes.

組織学でMRIをマージするために、3D印刷技術を活用：脳セクショニングのためのプロトコルを

Magnetic resonance imaging (MRI) allows for the delineation between normal and abnormal tissue on a macroscopic scale, sampling an entire tissue volume ...

Sublimation of DAN Matrix for the Detection and Visualization of Gangliosides in Rat Brain Tissue for MALDI Imaging Mass Spectrometry

Sample preparation is key for optimal detection and visualization of analytes in Matrix-assisted Laser Desorption/Ionization (MALDI) Imaging Mass Spectrometry (IMS) experiments. Determining the appropriate protocol to follow throughout the sample preparation process can be difficult as each step must be optimized to comply with the unique characteristics of the analytes of interest. This process involves not only finding a compatible matrix that can desorb and ionize the molecules of interest efficiently, but also selecting the appropriate matrix deposition technique. For example, a wet matrix deposition technique, which entails dissolving a matrix in solvent, is superior for desorption of most proteins and peptides, whereas dry matrix deposition techniques are particularly effective for ionization of lipids. Sublimation has been reported as a highly efficient method of dry matrix deposition for the detection of lipids in tissue by MALDI IMS due to the homogeneity of matrix crystal deposition and minimal analyte delocalization as compared to many wet deposition methods 1,2. Broadly, it involves placing a sample and powdered matrix in a vacuum-sealed chamber with the samples pressed against a cold surface. The apparatus is then lowered into a heated bath (sand or oil), resulting in sublimation of the powdered matrix onto the cooled tissue sample surface. Here we describe a sublimation protocol using 1,5-diaminonaphthalene (DAN) matrix for the detection and visualization of gangliosides in the rat brain using MALDI IMS.

A protocol for the sublimation of DAN matrix onto rat brain tissue for the detection of gangliosides using MALDI Imaging Mass Spectrometry is presented.

MALDIイメージングマススペクトロメトリーのためのラット脳組織におけるガングリオシドの検出と可視化のためのDANマトリックスの昇華

Sample preparation is key for optimal detection and visualization of analytes in Matrix-assisted Laser Desorption/Ionization (MALDI) Imaging Mass ...

Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the experimental time, and increases the number of animals that are utilized per study. This limitation specifically impacts protocols such as calcium imaging that require prolonged pre-incubation with bath-applied dyes. Exponential bacterial growth within 3 - 4 h after slicing is tightly correlated with a decrease in tissue health. This study describes a method for&#160;limiting the proliferation of bacteria in acute preparations to maintain viable neuronal tissue for prolonged periods of time (&#62;24 h) without the need for antibiotics, sterile procedures, or tissue culture media containing growth factors. By cycling the extracellular fluid through UV irradiation and keeping the tissue in a custom holding chamber at 15 - 16 &#176;C, the tissue shows no difference in electrophysiological properties, or calcium signaling through intracellular calcium dyes at &#62;24 h postdissection. These methods will not only extend experimental time for those using acute neuronal tissue, but will reduce the number of animals required to complete experimental goals, and will set a gold standard for acute neuronal tissue incubation.

Once removed from the body, neuronal tissue is greatly affected by environmental conditions, leading to eventual degradation of the tissue after 6 - 8 h. Using a unique incubation method, which closely monitors and regulates the extracellular environment of the tissue, tissue viability can be significantly extended for &#62;24 h.

電気生理学およびカルシウムイメージングのための急性神経組織の長期のインキュベーション

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the ...

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, enabling the use of D. melanogaster molecular genetics to study inositol-lipid signaling and Transient Receptor Potential (TRP) channels at the single-molecule level. A handful of labs have mastered this powerful technique, which enables the analysis of the physiological responses to light under highly controlled conditions. This technique allows control over the intracellular and extracellular media; the membrane voltage; and the fast application of pharmacological compounds, such as a variety of ionic or pH indicators, to the intra- and extracellular media. With an exceptionally high signal-to-noise ratio, this method enables the measurement of dark spontaneous and light-induced unitary currents (i.e.&#160;spontaneous and quantum bumps) and macroscopic Light-induced Currents (LIC) from single D. melanogaster photoreceptors. This protocol outlines, in great detail, all the key steps necessary to perform this technique, which includes both electrophysiological and optical recordings. The fly retina dissection procedure for the attainment of intact and viable ex vivo isolated ommatidia in the bath chamber is described. The equipment needed to perform whole-cell and fluorescence imaging measurements are also detailed. Finally, the pitfalls in using this delicate preparation during extended experiments are explained.

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell recordings from Drosophila melanogaster photoreceptors enable the measurement of spontaneous dark bumps, quantum bumps, macroscopic responses to light, and current-voltage relationships under various conditions. In combination with D. melanogaster genetic manipulation tools, this method enables the study of the ubiquitous inositol-lipid signaling pathway and its target, the TRP channel.

全細胞電圧クランプ記録のための電気生理学的方法<em&gt;ショウジョウバエ</em&gt;感光体

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, ...

Targeting Alpha Synuclein Aggregates in Cutaneous Peripheral Nerve Fibers by Free-floating Immunofluorescence Assay

To date, for most neurodegenerative diseases only a post-mortem histopathological definitive diagnosis is available. For Parkinson&#39;s disease (PD), the diagnosis still relies only on clinical signs of motor involvement that appear later on in the disease course, when most of the dopaminergic neurons are already lost. Hence, there is a strong need for a biomarker that can identify patients at the beginning of disease or at the risk of developing it. Over the last few years, skin biopsy has proved to be an excellent research and diagnostic tool for peripheral nerve diseases such as small fiber neuropathy. Interestingly, a small fiber neuropathy and alpha synuclein (&#945;Syn) neural deposits have been shown by skin biopsy in PD patients. Indeed, skin biopsy has the great advantage of being an easily accessible, minimally invasive and painless procedure that allows the analysis of peripheral nervous tissue prone to the pathology. Moreover, the possibility of repeating the skin biopsy in the course of the follow-up of the same patient allows studying the longitudinal correlation with the disease progression. We set up a standardized reliable protocol to investigate the presence of &#945;Syn aggregates in skin nerve fibers of the PD patient. This protocol involves few short fixation steps, a cryotome sectioning and then a free-floating immunofluorescence double-staining with two specific antibodies: anti Protein Gene Product 9.5 (PGP9.5) to mark the cutaneous nerve fibers and anti 5G4 for detecting &#945;Syn aggregates. It is a versatile, sensitive and easy to perform protocol that can also be applied for targeting other proteins of interest in skin nerves. The ability to mark &#945;Syn aggregates is another step forward to the use of skin biopsy as a tool for establishing a pre-mortem histopathological diagnosis of PD.

Here, we present a protocol for a free-floating indirect immunofluorescence assay on skin biopsy sections that allows for the identification of disease specific conformation variants of alpha synuclein involved in Parkinson disease and multiple proteins of the peripheral nervous system.

自由浮遊免疫蛍光アッセイによる皮下末梢神経線維におけるアルファシヌクレイン凝集体の標的化

To date, for most neurodegenerative diseases only a post-mortem histopathological definitive diagnosis is available. For Parkinson&#39;s disease (PD), the ...

A Hydrophobic Tissue Clearing Method for Rat Brain Tissue

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered tissue samples. Traditional immunolabeling procedures require cutting the sample into thin sections, which restricts the ability to label and examine intact structures. However, if brain tissue can remain intact during processing, structures and circuits can remain intact for the analysis. Previously established clearing methods take significant time to completely clear the tissue, and the harsh chemicals can often damage sensitive antibodies. The iDISCO method quickly and completely clears tissue, is compatible with many antibodies, and requires no special lab equipment. This technique was initially validated for the use in mice tissue, but the current protocol adapts this method to image hemispheres of control and transgenic rat brains. In addition to this, the present protocol also makes several adjustments to preexisting protocol to provide clearer images with less background staining. Antibodies for Iba-1 and tyrosine hydroxylase were validated in the HIV-1 transgenic rat and in F344/N control rats using the present hydrophobic tissue clearing method. The brain is an interwoven network, where structures work together more often than separately of one another. Analyzing the brain as a whole system as opposed to a combination of individual pieces is the greatest benefit of this whole brain clearing method.

Here we present a hydrophobic tissue clearing method that allows for the viewing of target molecules as part of intact brain structures. This technique has now been validated for F344/N control and HIV-1 transgenic rats of both sexes.

ラット脳組織の疎水性組織除去法

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered ...

Automated Gait Analysis to Assess Functional Recovery in Rodents with Peripheral Nerve or Spinal Cord Contusion Injury

Peripheral and central nerve injuries are mostly studied in rodents, especially rats, given the fact that these animal models are both cost-effective and a lot of comparative data has been published in the literature. This includes a multitude of assessment methods to study functional recovery following nerve injury and repair. Besides evaluation of nerve regeneration by means of histology, electrophysiology, and other in vivo and in vitro assessment techniques, functional recovery is the most important criterion to determine the degree of neural regeneration. Automated gait analysis allows recording of a vast quantity of gait-related parameters such as Paw Print Area and Paw Swing Speed as well as measures of inter-limb coordination. Additionally, the method provides digital data of the rats&#8217; paws after neuronal damage and during nerve regeneration, adding to our understanding of how peripheral and central nervous injuries affect their locomotor behavior. Besides the predominantly used sciatic nerve injury model, other models of peripheral nerve injury such as the femoral nerve can be studied by means of this method. In addition to injuries of the peripheral nervous systems, lesions of the central nervous system, e.g., spinal cord contusion can be evaluated. Valid and reproducible data assessment is strongly dependent on meticulous adjustment of the hard- and software settings prior to data acquisition. Additionally, proper training of the experimental animals is of crucial importance. This work aims to illustrate the use of computerized automated gait analysis to assess functional recovery in different animal models of peripheral nerve injury as well as spinal cord contusion injury. It also emphasizes the method&#8217;s limitations, e.g., evaluation of nerve regeneration in rats with sciatic nerve neurotmesis due to limited functional recovery. Therefore, this protocol is thought to help researchers interested in peripheral and central nervous injuries to assess functional recovery in rodent models.

Automated gait analysis is a feasible tool to evaluate functional recovery in rodent models of peripheral nerve injury and spinal cord contusion injury. While it requires only one setup to assess locomotor function in various experimental models, meticulous hard- and soft-ware adjustment and training of the animals is highly important.

末梢神経または脊髄損傷を伴うげっ歯類の機能回復を評価する自動歩行分析

Peripheral and central nerve injuries are mostly studied in rodents, especially rats, given the fact that these animal models are both cost-effective and ...