著者らは、フローサイトメトリーの支援をジェームズOesterlingに感謝したいと思います。この研究は、米国国立がん研究所（R01-CA178380、ZY）および米国国立衛生研究所博士研究訓練助成金（5T32CA009035-37、LWY）からの助成金によってサポートされていました。

このプロトコルにおける動物の使用は、フォックスチェイスがんセンター動物実験委員会によって承認された手順に従って行われている。 楽器、ソリューション、およびカバーガラスの作製<ol><li> ＃5細かい鉗子のクリーン二組、＃7の1組の罰金、湾曲鉗子、一つの大きな外科はさみ、1 microdissectingはさみ、1へら一あきスプーン。セルフシール滅菌袋とオートクレーブ中で楽器を置きます。使用するまで袋に楽器を保管してください。 </li><li> 3％の低融点アガロース溶液を調製する。 <ol><li>気泡が出現するまで、電子レンジ、暑さの中に置くの滅菌ダルベッコのリン酸緩衝生理食塩水（DPBS）50ml中に1.5グラムのアガロースを追加します。スワールフラスコと再加熱すべてアガロースを溶解するまで。塊が現れた場合、加熱前に攪拌棒を備えたソリューションを混ぜる。 </li><li> 37℃の水浴に保管ソリューションまで、必要に応じ。 </li><li>長期的なSTORAG​​用E、カ月間室温または4℃で、将来の使用のためにアガロース溶液を格納します。液化するために、電子レンジで再加熱する。アガロース溶液を繰り返し加熱するために、温かい滅菌蒸留水との初期重量を維持するために加熱する前に、フラスコの重量を量る。 </li></ol></li><li>以前に2に記載されているように、パパインベースの細胞解離のために以下の手段を準備します。 <ol><li>赤含有DPBSを滅菌フェノールでパパイン（100 U / ml）を、システイン（0.2 mg / mlで）およびDNase（250 U / ml）でパパイン溶液。 </li><li>ウシ血清アルブミンとオボムコイド溶液（BSA; 8 mg / ml）を、大豆トリプシンインヒビター（8 mg / ml）で）およびDNase（250 U / ml）の滅菌フェノールレッドを含有するDPBS中。 フィルターは両方のソリューションを殺菌し、フェノールレッドを含有するDPBSの元の色を返すようにpHを調整する。 </li></ol></li><li>細胞培養培地（NB / B27）を調製：1 mMピルビン酸ナトリウム、2mMのL-グルタミン、1％ペニシリン - ストレプトマイシン（P / S）及びB27サプリメントを基本培地を補う。 FilteR殺菌し、光から保護します。使用前に37℃で媒体を平衡化し、5％CO 2。 </li><li> FACS緩衝液を準備しDPBS中の5％ウシ胎児血清（FBS）を。 47.5ミリリットルのDPBSに2.5ミリリットルのFBSを追加します。 </li><li>ポリ-D-リジン（PDL）でコーティングされたカバースリップを準備します。 <ol><li>オートクレーブ新しいカバーガラス。 </li><li>コー​​トPDLとカバースリップ（滅菌蒸留水中に100μgの/ ml）を37℃で一晩または室温で2時間インキュベートする。 </li><li>ウォッシュは、滅菌蒸留水を用いてカバースリップ。 NB / B27培地で一回洗浄する。カバースリップは使用前に、組織培養フード内で完全に乾燥させます。 </li></ol></li></ol> 2解剖と組織スライスの調製<ol><li>解剖の日に、吸収パッドと、70％のエタノールとカバーで解剖領域を拭う。 </li><li>滅菌袋から解剖ツールを削除します。氷の上の小さなペトリ皿や場所に氷冷滅菌DPBS / 1％P / Sを追加します。 </li><li>首を切るP4 のMath1-GF大手術用ハサミでP / ネスチン-CFPの仔マウスを微湾曲した鉗子で頭を押したままにします。皮膚を外し、静かにへらを使って頭蓋骨から脳をかき出すとDPBS / 1％P / Sを含むペトリ皿に移すし、＃5細かい鉗子で頭蓋骨をはがす。 <ol><li> ＃5細かい鉗子を使用して、慎重に脳の他の部分から小脳を分離する。可能な限り迅速に一度に一つの子犬を解剖してください。 </li><li>分子·機能解析のための十分なNEPのを得るために、8小脳の最小値を収集します。 </li></ol></li><li> 3％低融点アガロース溶液で2×2×2cmの埋め込み型を満たす。アガロースの温度はせいぜい37℃ではないことを確認してください。 </li><li>実験室の組織を軽くたたくことにより、小脳から余分な液体を除去します。縦位置で金型内に小脳を置きます。それはすぐに固化するできるように、氷の上に金型を配置します。ブロックあたり〜4小脳を使用してください。 </li><li>李を取得振動ブレードビブラトームを用いて組織切片をヴィング。かみそりの刃で小脳を含むアガロースブ​​ロックをトリミング。垂直位置に配向小脳でビブラトーム板にアガロースブ​​ロックを接着。 </li><li>氷冷滅菌DPBS / 1％P / Sをビブラトームのトレイを記入し、下に氷を置く。 <ol><li>小脳の全長を通して600μmのセクションをカット。穴あきスプーンを使用して、製氷皿からの切片を収集します。氷上でDPBS / 1％P / Sで満たされたペトリ皿内の場所セクション。 </li></ol></li></ol> 3マイクロダイセクションおよび細胞解離<ol><li>蛍光解剖顕微鏡下で組織切片を含むペトリ皿を置きます。 <ol><li> CFP +分子層およびGFP + EGL（ 図1の点線を参照）との間での解剖による細かい鉗子を用いて小脳セクションの残りの部分からEGLを慎重に分離。になり、これ分子層の任意の部分を含まないように注意してくださいネスチン発現バーグマングリアの汚染。 </li><li>可能な限り迅速に、この手順を完了し、細胞死を回避するために、氷上で組織を維持する。 </li><li>次のステップに進む前に、周囲の組織アガロースを削除します。 </li></ol></li><li>以前に、単一細胞懸濁液を得るために、2に記載のようにパパインベースのプロトコルを使用して顕微解剖EGL組織を消化する。 <ol><li>再懸濁FACSを介して、CFP-陽性細胞の収集のためのFACS緩衝液で細胞を。 </li></ol></li></ol> 4。セルソーティングおよびめっき<ol><li>適切なCFPフィルターを含む滅菌、高速フローサイトメーターを使用して、CFP蛍光のソート細胞と氷上で、FACS緩衝液で細胞を集める。各P4小脳からの約10万のNEPを取得します。 </li><li> 5分間300×gで遠心分離した細胞。さらなる実験のための分子解析や培養のために、すぐに細胞を使用してください。 </li><li>培養細胞に、予め温めておいたNB / B27 mの細胞を再懸濁以前に2に記載されているように、PDLコーティングしたカバーガラス上EDIAとプレート。 </li></ol>

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著者らは、開示することは何もない。

ここで説明顕微解剖法は、分子的および機能的分析において使用するための生細胞の純粋な集団を単離することができる最初の手順である。 Li ら9によって実証されるように、 この方法で得られたのNEPを培養し、各種実験に組み込むことができ、その高い純度で、マイクロアレイ分析のためにも使用することができる。 これは顕微解剖技術に習熟?...

小脳は、それぞれが異なる細胞型を含む、複数の細胞層から構成されている。開発中は、EGLは分子層とプルキンエ層はそれぞれ、バーグマングリアとプルキンエニューロンを含み、一方顆粒ニューロン前駆体（のGNP）が増殖して含まれています。小脳内のディープは、神経幹細胞（NSC）およびオリゴデンドロサイト1が含まれている白質を、ある。 ソニックヘッジホッグ（Shhが）小脳発生の調節において重要な役割を果たしており、特に、パッチが適用その受容体（PTC）、Shhの2-4シグナルの負の調節因子への結合を介してのGNPの増殖を促進する。 Shhシグナル伝達の異常な活性化は、髄芽腫（MB）、小児5,6で最も一般的な悪性脳腫瘍を生成します。 PTCミュータントMBトランスジェニックマウスモデルは、MBの研究のための強力なツールであり、人間のMB 7,8に似た腫瘍を発症。これらを使用したマウスは、それがのGNPはハリネズミ型MB 7起源の細胞であることが発見された。さらに、のG​​NPに加えて、私たちは最近、またMBを生じさせることができる開発する小脳のEGL内の神経前駆細胞のユニークな集団を同定した。これらの細胞は、タイプVIの中間フィラメントタンパク質、ネスチンを高レベルで発現し、9のNEPと呼ばれる。のNEPは、EGLの深部内に配置され、唯一の新生児小脳の開発中に一過的に存在している。彼らはのGNPのような顆粒細胞系譜にコミットしますが、彼 ​​らは静止状態にしてのMath1、従来のGNP 10のためのよく確立されたマーカーを発現しないように区別されます。さらに、NEPのは彼らMBの腫瘍形成のための新規の原点になりShhのシグナル伝達経路9、起動後のMBの上昇よりも効率的にのGNPを与える。 私たちは、以前に顕微解剖技術によって生後4日目（P4）での小脳のEGLからのNEPを隔離ここで9について説明した。組織の直接顕微鏡可視化を介したマイクロダイセクションは、小脳のEGLの特定の解剖を可能にします。ネスチンはまた、分子層1,7,11,12に小脳白質中のNSCによってとバーグマングリアによって発現し、それは彼らが分析を混乱さと同じように、これらの細胞は、含まれていないことが非常に重要でしているように、これが必要です。顕微解剖組織から単離された細胞は、分子分析のために直ちに使用することができ、またはそれらは、さらなる用途のために培養することができる。 具体的にはEGLをmicrodissectingにおいて補助するために、さらなるのNEPを精製する、 のMath1-GFP（緑色蛍光タンパク質）マウスは、 ネスチン-CFP（シアン蛍光タンパク質）マウスと交配した。 Math1または特異的のGNP及びGFPの発現によって発現される転写因子は、EGL 9,13においてはっきりと見える。 ネスチン-CFPマウスはCFPの核型を発現し、容易に分子層9,14に視覚化することができる。一緒に、GFPとCFP式がEGLのマイクロダイセクションのための境界を作成します（ 図1を参照）。 CFP陽性のNEPは、その後解剖しEGLsの酵素的解離に続いて、FACSによって単離した。 現在、EGLから細胞を単離するための最もよく利用される方法は、全小脳組織15,16のパーコール勾配遠心分離である。しかし、この方法は、完全に分子層と白質のネスチン+細胞集団を除外することができないのでのNEPの研究のために使用することができない。関心のある細胞を除去するためにレーザエネルギーの転送を利用するレーザーキャプチャーマイクロダイセクションは、異種の組織17,18内の特定の細胞を単離するために使用されるが、捕捉された細胞のみがDNA、RNAおよびタンパク質の回収のために使用されないことができる別の方法である培養することができるように。 このプロトコルは、具体的には、EGLからのNEPの生きている、純粋な集団を単離する方法を提供します。この技術は、認識可能な解剖学的アーキテクチャまたは蛍光標識された細胞/領域のいずれかを有する異なる組織タイプに適用することができる。したがって、細胞単離プロトコールにこの新規顕微解剖技術を組み込むの主な利点は、細胞が隣接する組織を汚染除去するために、特定の組織領域から単離することができ、細胞は、他のアプリケーションの分析または培養のためにすぐに収集することができることである。

<table border="0" cellpadding="0" cellspacing="0" width="572">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Neurobasal media</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">21103-049</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">PDL</td>
			<td style="width:71px;">Millipore</td>
			<td style="width:119px;">A-003-E</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">ultra pure low melting temperature agarose</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">16520-050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">DPBS</td>
			<td style="width:71px;">Gibco</td>
			<td align="right" style="width:119px;">14190144</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of distinct cell populations from the developing cerebellum by microdissection

顕微解剖は、組織の特定の領域を分離し、隣接領域における細胞源からの汚染を排除することができる新規な技術である。この方法は、最初にネスチン発現前駆細胞（のNEP）の研究では、小脳の外部胚層（EGL）内の細胞の新たに同定された集団において利用した。蛍光活性化細胞分類（FACS）と組み合わせてマイクロダイセクションを使用して、のNEPの純粋な集団は、小脳におけるEGLにおける従来の顆粒神経細胞前駆体から及び他の汚染ネスチン発現細胞とは別に収集した。マイクロダイセクションがなければ、のNEPの機能解析は、パーコール勾配遠心分離およびレーザーキャプチャーマイクロダイセクションとして利用可能な現在の方法では不可能でした。この技術は、認識可能な領域または蛍光標識された細胞のいずれかを含むさまざまな組織との使用のために適用することができる。最も重要なことは、このmicrodissectioの主な利点n個の技術は、単離された細胞が生きていると、他に記載された方法で、現在は不可能であるさらなる実験のために培養することができるということです。

図1Aに示すように、小脳スライスは小脳EGLは、GFP発現のGNPによって支配された分子層がCFP陽性グリア細胞によって濃縮された、P4 のMath1-GFP / ネスチン-CFPマウスから調製した。 （; 図1A点線に沿って）EGLの深部に位置しているのNEPを単離するために、スライスは、EGLと分子層の間に顕微解剖された。解剖EGLs（ 図1B）は、次いで、FACS続い酵素?...

Watch this Scientific Journal Video about Isolation of Distinct Cell Populations from the Developing Cerebellum by Microdissection at JoVE.com

Isolation of Distinct Cell Populations from the Developing Cerebellum by Microdissection

ネスチン発現前駆細胞は、発展途上小脳における神経前駆細胞の新たに同定された集団である。蛍光活性化細胞選別と組み合わせてここで示さ顕微解剖技術を用いて、この細胞集団は、他の小脳領域から汚染を含んでいない精製することができ、さらなる研究のために培養することができる。

マイクロダイセクションによる開発小脳とは異なる細胞集団の単離

Microdissection is a novel technique that can isolate specific regions of a tissue and eliminate contamination from cellular sources in adjacent areas. ...

isolation-distinct-cell-populations-from-developing-cerebellum

Research

JoVE Journal

Neuroscience

9.5K ビュー.  Temple University Health System. ネスチン発現前駆細胞は、発展途上小脳における神経前駆細胞の新たに同定された集団である。蛍光活性化細胞選別と組み合わせてここで示さ顕微解剖技術を用いて、この細胞集団は、他の小脳領域から汚染を含んでいない精製することができ、さらなる研究のために培養することができる。 

ビデオ: Isolation of Distinct Cell Populations from the Developing Cerebellum by Microdissection

Electrophysiological Characterization of GFP-Expressing Cell Populations in the Intact Retina

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack conspicuous morphological features or show a low population density. This applies particularly to retinal amacrine cells, an exceptionally multiform class of interneurons that comprise roughly 30 subtypes in mammals1. Though being a crucial part of the visual processing by shaping the retinal output2, most of these subtypes have not been studied up to now in a functional context because encountering these cells with a recording electrode is a rare event.
Recently, a multitude of transgenic mouse lines is available that express fluorescent markers like green fluorescent protein (GFP) under the control of promoters for membrane receptors or enzymes that are specific to only a subset of neurons in a given tissue3,4. These pre-labeled cells are therefore accessible to directed microelectrode targeting under microscopic control, permitting the systematic study of their physiological properties in situ. However, excitation of fluorescent markers is accompanied by the risk of phototoxicity for the living tissue. In the retina, this approach is additionally hampered by the problem that excitation light causes appropriate stimulation of the photoreceptors, thus inflicting photopigment bleaching and transferring the retinal circuits into a light-adapted condition. These drawbacks are overcome by using infrared excitation delivered by a mode-locked laser in short pulses of the femtosecond range. Two-photon excitation provides energy sufficient for fluorophore excitation and at the same time restricts the excitation to a small tissue volume minimizing the hazards of photodamage5. Also, it leaves the retina responsive to visual stimuli since infrared light (&gt;850 nm) is only poorly absorbed by photopigments6.
In this article we demonstrate the use of a transgenic mouse retina to attain electrophysiological in situ recordings from GFP-expressing cells that are visually targeted by two-photon excitation. The retina is prepared and maintained in darkness and can be subjected to optical stimuli which are projected through the condenser of the microscope (Figure 1). Patch-clamp recording of light responses can be combined with dye filling to reveal the morphology and to check for gap junction-mediated dye coupling to neighboring cells, so that the target cell can by studied on different experimental levels.

This article depicts the recording of individual cells from fluorescently tagged neuronal populations in the intact mouse retina. By using two-photon infrared excitation transgenetically labeled cells were targeted for patch-clamp recording to study their light responses, receptive field properties, and morphology.

無傷の網膜におけるGFP発現細胞集団の電気生理学的特性

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack ...

神経科学

Laser Capture Microdissection of Enriched Populations of Neurons or Single Neurons for Gene Expression Analysis After Traumatic Brain Injury

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that is critical for learning, memory and executive function.1,2 Hence our studies focus on gene expression analysis of specific neuronal populations in distinct subregions of the hippocampus. The technique of laser capture microdissection (LCM), introduced in 1996 by Emmert-Buck, et al.,3 has allowed for significant advances in gene expression analysis of single cells and enriched populations of cells from heterogeneous tissues such as the mammalian brain that contains thousands of functional cell types.4 We use LCM and a well established rat model of traumatic brain injury (TBI) to investigate the molecular mechanisms that underlie the pathogenesis of TBI. Following fluid-percussion TBI, brains are removed at pre-determined times post-injury, immediately frozen on dry ice, and prepared for sectioning in a cryostat. The rat brains can be embedded in OCT and sectioned immediately, or stored several months at -80 &deg;C before sectioning for laser capture microdissection. Additionally, we use LCM to study the effects of TBI on circadian rhythms. For this, we capture neurons from the suprachiasmatic nuclei that contain the master clock of the mammalian brain. Here, we demonstrate the use of LCM to obtain single identified neurons (injured and degenerating, Fluoro-Jade-positive, or uninjured, Fluoro-Jade-negative) and enriched populations of hippocampal neurons for subsequent gene expression analysis by real time PCR and/or whole-genome microarrays. These LCM-enabled studies have revealed that the selective vulnerability of anatomically distinct regions of the rat hippocampus are reflected in the different gene expression profiles of different populations of neurons obtained by LCM from these distinct regions. The results from our single-cell studies, where we compare the transcriptional profiles of dying and adjacent surviving hippocampal neurons, suggest the existence of a cell survival rheostat that regulates cell death and survival after TBI.

We describe how to use laser capture microdissection (LCM) to obtain enriched populations of hippocampal neurons or single neurons from frozen sections of the injured rat brain for subsequent gene expression analysis using quantitative real time PCR and/or whole-genome microarrays.

外傷性脳損傷後の神経細胞や遺伝子発現解析のための単一ニューロンの濃縮された集団のレーザーキャプチャーマイクロダイセクション

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that ...

Production of RNA for Transcriptomic Analysis from Mouse Spinal Cord Motor Neuron Cell Bodies by Laser Capture Microdissection

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or between the same cell type in health and disease or following pharmacologic treatments.&#160;In the spinal cord, such preparation from motor neurons, the target of interest in many neurologic and neurodegenerative diseases, is complicated by the fact that motor neurons represent &#60;10% of the total cell population.&#160;Laser capture microdissection (LMD) has been developed to address this problem.&#160;Here, we describe a protocol to quickly recover, freeze, and section mouse spinal cord to avoid RNA damage by endogenous and exogenous RNases, followed by staining with Azure B in 70% ethanol to identify the motor neurons while keeping endogenous RNase inhibited.&#160;LMD is then used to capture the stained neurons directly into guanidine thiocyanate lysis buffer, maintaining RNA integrity.&#160;Standard techniques are used to recover the total RNA and measure its integrity.&#160;This material can then be used for downstream analysis of the transcripts by RNA-seq and qRT-PCR.

High-quality total RNA has been prepared from cell bodies of mouse spinal cord motor neurons by laser capture microdissection after staining spinal cord sections with Azure B in 70% ethanol.&#160;Sufficient RNA (~40-60 ng) is recovered from 3,000-4,000 motor neurons to allow downstream RNA analysis by RNA-seq and qRT-PCR.

レーザー捕捉マイクロ解離によるマウス脊髄運動ニューロン細胞体からの転写解析のためのRNAの生成

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

発生中のマウス胚性皮質における有糸分裂のライブイメージング

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Laser Capture Microdissection - A Demonstration of the Isolation of Individual Dopamine Neurons and the Entire Ventral Tegmental Area

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue sections on a microscope slide. When combined with immunohistochemistry, LCM can be used to isolate individual cells types based on a specific protein marker. Here, the LCM technique is described for collecting a specific population of dopamine neurons directly labeled with&#160;tyrosine hydroxylase immunohistochemistry and for isolation of the dopamine neuron containing region of the ventral tegmental area using indirect tyrosine hydroxylase immunohistochemistry on a section adjacent to those used for LCM. An infrared (IR) capture laser is used to both dissect individual neurons as well as the ventral tegmental area off glass slides and onto an LCM cap for analysis. Complete dehydration of the tissue with 100% ethanol and xylene is critical. The combination of the IR capture laser and the ultraviolet (UV) cutting laser is used to isolate individual dopamine neurons or the ventral tegmental area when using PEN membrane slides. A PEN membrane slide has significant advantages over a glass slide as it offers better consistency in capturing and collecting cells, is faster collecting large pieces of tissue, is less reliant on dehydration and results in complete removal of the tissue from the slide. Although removal of large areas of tissue from a glass slide is feasible, it is considerably more time consuming and frequently leaves some residual tissue behind. Data shown here demonstrate that RNA of sufficient quantity and quality can be obtained using these procedures for quantitative PCR measurements. Although RNA and DNA are the most commonly isolated molecules from tissue and cells collected with LCM, isolation and measurement of microRNA, protein and epigenetic changes in DNA can also benefit from the enhanced anatomical and cellular resolution obtained using LCM.

The isolation of individual dopamine neurons or the ventral tegmental area with direct or indirect immunohistochemistry is demonstrated using laser capture microdissection. Parameters for isolation of tissue from a glass slide using an infrared laser and from membrane slides using the combination of an infrared and ultraviolet laser are discussed.

レーザーキャプチャーマイクロダイセクション - 個々のドーパミンニューロンの単離と全体の腹側被蓋領域の実証

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue ...

In-depth Physiological Analysis of Defined Cell Populations in Acute Tissue Slices of the Mouse Vomeronasal Organ

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory neurons (VSNs) express a specific type of G protein-coupled receptor (GPCR) from at least three different chemoreceptor gene families allowing sensitive and specific detection of chemosensory cues. These families comprise the V1r and V2r gene families as well as the formyl peptide receptor (FPR)-related sequence (Fpr-rs) family of putative chemoreceptor genes. In order to understand the physiology of vomeronasal receptor-ligand interactions and downstream signaling, it is essential to identify the biophysical properties inherent to each specific class of VSNs.
The physiological approach described here allows identification and in-depth analysis of a defined population of sensory neurons using a transgenic mouse line (Fpr-rs3-i-Venus). The use of this protocol, however, is not restricted to this specific line and thus can easily be extended to other genetically modified lines or wild type animals.

Here, we describe a physiological approach that allows identification and in-depth analysis of a defined population of sensory neurons in acute coronal tissue slices of the mouse vomeronasal organ using whole-cell patch-clamp recordings.

マウス鋤鼻器官の急性組織スライスで定義された細胞集団の中で、深さ生理学的解析

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory ...

Live Imaging Followed by Single Cell Tracking to Monitor Cell Biology and the Lineage Progression of Multiple Neural Populations

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate decisions, will be crucial to design therapeutic strategies for many diseases affecting the nervous system. Current methods to track cell populations rely on their final outcomes in still images and they generally fail to provide sufficient temporal resolution to identify behavioral features in single cells. Moreover, variations in cell death, behavioral heterogeneity within a cell population, dilution, spreading, or the low efficiency of the markers used to analyze cells are all important handicaps that will lead to incomplete or incorrect read-outs of the results. Conversely, performing live imaging and single cell tracking under appropriate conditions represents a powerful tool to monitor each of these events. Here, a time-lapse video-microscopy protocol, followed by post-processing, is described to track neural populations with single cell resolution, employing specific software. The methods described enable researchers to address essential questions regarding the cell biology and lineage progression of distinct neural populations.

A robust protocol to monitor neural populations by time-lapse video-microscopy followed by software-based post-processing is described. This method represents a powerful tool to identify biological events in a selected population during live imaging experiments.

ライブ イメージング モニター細胞生物学および複数の神経集団の血統で、進行を追跡 1 つのセルに続いて

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate ...

Optimization of Laser-Capture Microdissection for the Isolation of Enteric Ganglia from Fresh-Frozen Human Tissue

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). We have identified five steps in the workflow that are crucial for obtaining RNA isolates from enteric ganglia with sufficiently high quality and quantity for RNA-seq. First, when preparing intestinal tissue, each sample must have all excess liquid removed by blotting prior to flattening the serosa as much as possible across the bottom of large base molds. Samples are then quickly frozen atop a slurry of dry ice and 2-methylbutane. Second, when sectioning the tissue, it is important to position cryomolds so that intestinal sections parallel the full plane of the myenteric plexus, thereby yielding the greatest surface area of enteric ganglia per slide. Third, during LCM, polyethylene napthalate (PEN)-membrane slides offer the greatest speed and flexibility in outlining the non-uniform shapes of enteric ganglia when collecting enteric ganglia. Fourth, for distinct visualization of enteric ganglia within sections, ethanol-compatible dyes, like Cresyl Violet, offer excellent preservation of RNA integrity relative to aqueous dyes. Finally, for the extraction of RNA from captured ganglia, we observed differences between commercial RNA extraction kits that yielded superior RNA quantity and quality, while eliminating DNA contamination. Optimization of these factors in the current protocol greatly accelerates the workflow and yields enteric ganglia samples with exceptional RNA quality and quantity.

The goal of this protocol is to obtain high-integrity RNA samples from enteric ganglia isolated from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). This protocol involves preparing flash-frozen samples of human intestinal tissue, cryosectioning, ethanolic staining and dehydration, LCM, and RNA extraction.

新鮮凍結ひと組織から腸管神経節の隔離のためのレーザー キャプチャ レーザーマイクロダイ セクションの最適化

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue ...

A Stainless Protocol for High Quality RNA Isolation from Laser Capture Microdissected Purkinje Cells in the Human Post-Mortem Cerebellum

Laser capture microdissection (LCM) is an advantageous tool that allows for the collection of cytologically and/or phenotypically relevant cells or regions from heterogenous tissues. Captured product can be used in a variety of molecular methods for protein, DNA or RNA isolation. However, preservation of RNA from postmortem human brain tissue is especially challenging. Standard visualization techniques for LCM require histologic or immunohistochemical staining procedures that can further degrade RNA. Therefore, we designed a stainless protocol for visualization in LCM with the intended purpose of preserving RNA integrity in post-mortem human brain tissue. The Purkinje cell of the cerebellum is a good candidate for stainless visualization, due to its size and characteristic location. The cerebellar cortex has distinct layers that differ in cell density, making them a good archetype to identify under high magnification microscopy. Purkinje cells are large neurons situated between the granule cell layer, which is a densely cellular network of small neurons, and the molecular layer, which is sparse in cell bodies. Because of this architecture, the use of stainless visualization is feasible. Other organ or cell systems that mimic this phenotype would also be suitable. The stainless protocol is designed to fix fresh-frozen tissue with ethanol and remove lipids with xylene for improved morphological visualization under high magnification light microscopy. This protocol does not account for other fixation methods and is specifically designed for fresh-frozen tissue samples captured using an ultraviolet (UV)-LCM system. Here, we present a full protocol for sectioning and fixing fresh frozen post-mortem human cerebellar tissue and purification of RNA from Purkinje cells isolated by UV-LCM, while preserving RNA quality for subsequent RNA-sequencing. In our hands, this protocol produces exceptional levels of cellular visualization without the need for staining reagents and yields RNA with high RNA integrity numbers (&#8805;8) as needed for transcriptional profiling experiments.

This protocol uses a stain-free approach to visualize and isolate Purkinje cells in fresh-frozen tissue from human post-mortem cerebellum via laser capture microdissection. The purpose of this protocol is to generate sufficient amounts of high-quality RNA for RNA-sequencing.

ステンレス高品質レーザーからの RNA の隔離のプロトコルをキャプチャ人間死後小脳 Microdissected プルキンエ細胞

Laser capture microdissection (LCM) is an advantageous tool that allows for the collection of cytologically and/or phenotypically relevant cells or ...

Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model system is to provide a controlled microenvironment and maintain the high survival rate and the natural features of dissociated neuronal and nonneuronal cells as much as possible under in vitro conditions. In this article, we demonstrate a method of isolating primary neurons from the developing mouse cerebellum, placing them in an in vitro environment, establishing their growth, and monitoring their viability and differentiation for several weeks. This method is applicable to embryonic neurons dissociated from cerebellum between embryonic days 12&ndash;18.

Conducting in vitro experiments to reflect in vivo conditions as adequately as possible is not an easy task. The use of primary cell cultures is an important step toward understanding cell biology in a whole organism. The provided protocol outlines how to successfully grow and culture embryonic mouse cerebellar neurons.

胚性マウス小脳から単離されたニューロンの一次培養

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model ...