著者は、洞察力に富んだ議論や提案のためのTrkA taulacZマウスと博士アネットマルクス博士ルイライハルトに感謝したいと思います。この作品はバーク財団だけでなく、ホワイトホール財団研究助成金2010-08-61、ライフ財団（WFL-US-028/14）のための翼から研究助成金からスタートアップ資金によってサポートされていましたから、ZB1-1102-1を付与クリストファー＆ダナ·リーブ財団、国立眼研究所から、JZへの助成金1R01EY022409と3R01EY022409-01S1。 KJOはゴールドスミスの仲間です。

注：すべての手順は、実験動物の使用とケアのためのNIHガイドに準拠しています。動物プロトコルは、ワイルコーネル医科大学のIACUCによって承認された。 1.組織調製<ol><li>頸椎脱臼15によって時限妊娠女性を安楽死させる。冷リン酸緩衝生理食塩水（PBS）を充填した6ウェルディッシュのウェルで個々に時限妊娠雌場所胚からE18胚 - 胚E16を解剖。 </li><li>冷PBSで胚を洗浄します。 </li><li>その後の遺伝子型判定のための事前準備されたプロテイナーゼK溶液中で胚と場所の羊膜のわずかな部分を削除します。羊膜が失われた場合あるいは、胚の尾の先端の一部を除去し、プロテイナーゼK溶液に入れ</li><li>虫ピン（側面当たり少なくとも100）を使用して、すべての胚の周りの皮膚に穴を突く。 </li><li> PBSを除去し、ゆっくりと新鮮な2％パラホルムアルデヒド（PFA）を追加し、ウェルにpHが7.4、。 </li><li>ゆっくりと4℃で2時間振る。 </li><li> PBSで2回胚をすすぎ、染色まで4℃でPBS中に保管してください。 </li></ol> 2.ジェノタイピング<ol><li>製造業者の説明書に従って、プロテイナーゼK混合物中の羊膜または胚の尾部を浸す。 </li><li> 10分間56℃でインキュベートする。 </li><li>市販のDNA精製キットを用いてDNAを抽出する。 </li><li>遺伝子特異的なセンスおよびアンチセンスプライマー（10μM）の各0.5μlの、2μLのdNTPミックスとそれを組み合わせる、ゲノムDNA溶液に精製合計200μLの0.5μLを取る（のdATP 2.5mMの、dCTP、dGTPおよびdTTP） 、0.2μlのTaqポリメラーゼ（5U /μL）と2μlのPCR緩衝液（10倍）は、20μlの総体積にH 2 Oを追加します。 注：プライマー配列は以下のとおりである（ 表1）： のTrkA：TrkA_F：GCG GGC GCC GCC、GCG ATG、TrkA_R：GAA GCC GCC TGC GCG GCT CTG CCA GGG TG。 PCRのFRAGメントの長さ：400塩基対（bp）の。 バックス：In5R：GTT GAC CAG AGT GGC GTA GG、Ex5F：TGA TCA GAA CCA TCA TG、のNeoR：CCG CTT CCA TTG CTC AGC GG。 PCR断片の長さ：野生型、304塩基対;のBaxヌル、507 bpの。 TauLacZ：lacZup：ACA ACG TCG TGA GTG GGA AAA、lacZdown：ATC AAC ATT AAA TGT GAG CGA G。 PCR断片の長さ：360塩基対。 </li><li> TrkAのための以下のプログラムでPCRを実行します。72℃で30秒：68℃、ステップ4で20秒：ステップ3 95℃、10秒：ステップ1：ステップ2 95℃、1分。 40サイクル4 - を繰り返して、2を繰り返します。 <ol><li> LacZおよびBaxのために、以下のプログラムを用いてPCRを実行します。手順1：1分95℃​​で、ステップ2：10秒95℃、ステップ3：1分54℃、ステップ4：72で1分35サイクル4 - °Cを繰り返し、2を繰り返します。 </li></ol></li><li> DNAラダーのレーンで20分間、1×トリス - 酢酸-EDTA緩衝液中、100Vで2％アガロースゲル上で実行PCRサンプルフラグメントの長さを評価する。 </li><li>野生型のTrkA、Tの存在のための胚を分析RKA-tauLacZおよびBaxヌル対立遺伝子。実験的な胚の遺伝子型は、TrkAのtaulacZ / WTです：バックス- / -およびTrkAのtaulacZ / taulacZ：バックス- / - 、実験的な質問に応じて。 tauLacZレポーターを欠いている胚は軸索染色について陰性であることになるので、染色プロセスを較正するために陰性対照として役立つことができる。 </li></ol> 3.胚染色<ol><li> 3.8 -セクション3.2で下記のようにいくつかの変更を加えてLacZ染色のための市販のキットを使用してください。ここでは、ミリポアキットを使用。 </li><li>室温で穏やかに揺れ、少なくとも1時間緩衝液A中の胚を浸し。 </li><li>直接室温で静かに揺れ、少なくとも15分間、緩衝液Bで胚を浸す。 </li><li> 37℃での胚がバッファーA / Bであるが、前加温組織ベースのソリューション。胚あたりの組織塩基溶液の5ミリリットルを使用してください。 </li><li>胚は緩衝液A / Bにある間、新鮮な5-BRを準備100％のジメチルスルホキシド（DMSO）中に40mg / mlの濃度でomo-が4-クロロ-3-インドリルα-D-ガラクトピラノシド（X-galで）。 </li><li> 1 mg / mlの濃度で予め温め組織ベース溶液中にX-galで希釈し、ガラスシンチレーションバイアルにX-galを/組織ベース混合物を5ml加える。 </li><li> X-galを/組織ベースの混合物を含むガラスシンチレーションバイアルに胚を移す。パラフィルムで蓋を密封し、37℃で一晩インキュベート、ブルーステインの存在をチェックする。 </li><li>新鮮な4％PFA、pHが7.4とPostfixの胚。静かに、4℃で4時間振る。 </li><li>冷たいPBSで2回の胚をすすぐ。 </li></ol> 4.組織脱水とクリア<ol><li>ガラスバイアル中で振盪し、室温で30分間、50％メタノール/ 50％PBS混合物中の場所胚。 </li><li>ガラスバイアル中で振盪し、室温で30分間、75％メタノール/ 25％PBSで脱水し続ける。 </li><li> P 10パーセント/ 90％メタノール中で脱水し続けるガラスバイアル中で振盪し、室温で30分間、BS、。 </li><li>ガラスバイアル中で振盪し、室温で30分間（2×）、100％メタノール中で脱水し続ける。 </li><li> 1（V：V）ベンゾイルアルコール及びベンゾイル安息香酸の混合胚は、脱水されていますが、1を準備します。 注：ベンゾイルアルコール、ベンゾイル安息香酸は有毒である。 </li><li> 1：1で胚を浸す（v：v）によって精製した100％メタノールの混合物：15分間、100％BABB。 BABBはプラスチックを溶解するので、唯一のガラスを使用しています。 </li><li>完全に組織をクリアするとクリア胚におけるX-gal染色を視覚化するために100パーセントBABBで胚を浸し。 注：できるだけ早く画像のX-gal染色は、時間をかけてフェードインしますので。 </li></ol> 5.ホールマウントイメージング<ol><li>金属ピンセットを用いて写真撮影のために適切な角度で、100パーセントBABBに浸漬胚を、安定化させる。ライトアップアンダー光光源の組み合わせを用いて照明する。 </li><li>解剖顕微鏡で画像を取得するデジタルカメラを装備。 0.5ミリメートル離れてZ軸に沿って連続する5つの焦点面に複数の明視野エクスポージャーを取る。露光時間とFストップは、照明とカメラのスペックに応じて変化し、それぞれのセットアップのために最適化する必要があるかもしれません。 </li><li>オーバーレイ画像を処理し、フォトモンタージュを生成するために、標準的な画像処理ソフトウェアを使用してください。 </li></ol>

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The authors declare that there are no competing interests.

胚のTrkA taulacZマウスの上述のX-gal染色手順はそのまま固定胚における長距離軸索突起の迅速で詳細な視覚化を可能にする。 Baxのヌルバックグラウンドのため、これらのマウスは軸索成長とニューロンの生存の両方に寄与し得るシグナル伝達機構のプロービングが可能になります。興味のあるトランスジェニックまたはノックアウトマウスと交配すると、軸...

正確な神経回路網の確立は、神経系の機能性に不可欠な複雑な発達過程である。このプロセスの乱れは、ヒトの神経系疾患1-3に関与している神経細胞の機能障害を引き起こす。哺乳動物における軸索成長および標的神経支配の下にある分子メカニズムを研究するために、我々は2つ​​の遺伝的に改変されたマウス系統の組み合わせを使用して感覚ニューロンのTrkA発現の軸索の軌道を視覚化するためのプロトコルを開発した。 のTrkAは、神経成長因子NGFに対する受容体であり、侵害受容感覚ニューロン4の機能的マーカーである。 TrkAのは非常に初期の開発中に侵害受容ニューロンに発現し、NGF依存ニューロンの生存、軸索成長、樹枝状分岐とターゲット神経支配5-9を媒介する。 TrkAのtaulacZマウスでは、野生型のTrkA遺伝子taulacZ式 cで置換されている推定上のTrkA陽性ニューロンの軸索の形態11染色のβ-gal（X-galで）によって可視化することができるようassette 10、。ヘテロ接合のTrkA taulacZ / WTラインを使用して、我々は調節またはin vivoでの感覚求心性突出部の発展を妨害する可能性のある要因を調べることができます。 また、TrkAの発現は、したがって、NGF / TrkAのシグナル伝達の非存在下でのメカニズムを促進する軸索成長を評価するために使用することができるホモ接合性のTrkA taulacZ / taulacZのマウスには存在しない。侵害受容ニューロンは、NGF / TrkAのだけでなく、軸索成長のためのシグナリングに依存するためではなく、生存のために、我々は、それ以外の細胞死からそれらを救出、胚DRGニューロンにおけるアポトーシスを阻害するために、プロアポトーシスBax遺伝子を欠いている、別のマウス系統を使用するTrkAレセプターシグナル伝達の非存在下で観察。 バックス- / -バックグラウンド12は、このように molecuが可能に特に軸索成長7-9,13-15に影響を与えるシグナル伝達経路のLAR解剖。 TrkAのでは- / - ：バックス- / -マウスは、DRGニューロンは生き残るが、皮膚の感覚求心性神経支配は完全14,15を廃止している。私たちは、選択的に軸索投射の発展にそれぞれの貢献を決定するシグナル伝達経路を活性化することができる。 バックス- / -背景： - - /またはTrkA taulacZ / WTバックス：この方法の有用性は、異なる遺伝的改変はTrkAのtaulacZ / taulacZに飼育されているときに、軸索成長表現型の変化の評価を可能にすることである。

<table border="0" cellpadding="0" cellspacing="0" width="465">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:199px;"></td>
			<td style="width:127px;">Company</td>
			<td style="width:139px;">Catalog Number</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PFA</td>
			<td>Sigma-Aldrich</td>
			<td>P6418</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td>Life Tech</td>
			<td>10010-023</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue Rinse Solution A</td>
			<td>Millipore</td>
			<td>BG-6-B</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue Rinse Solution B</td>
			<td>Millipore</td>
			<td>BG-7-B</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue Stain Base Solution</td>
			<td>Millipore</td>
			<td>BG-8-C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">X-gal&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>B4252</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass scintiallation vial</td>
			<td>Kimble Chase</td>
			<td>74500-20</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Incubator</td>
			<td>Labline</td>
			<td>Model 120</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insect pins</td>
			<td>FST</td>
			<td>26000-30</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D8418</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6 well dish</td>
			<td>USA Scientific</td>
			<td>CC7672-7506</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Primers</td>
			<td>IDT</td>
			<td>custom DNA primers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Takara dNTP mixture</td>
			<td>Takara</td>
			<td>4030</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Takara LA buffer</td>
			<td>Takara</td>
			<td>RR002A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Takara LA Taq</td>
			<td>Takara</td>
			<td>RR002A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PCR machine</td>
			<td>Bio-Rad&#160;</td>
			<td>DNA Engine Dyad</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Benzyl alcohol</td>
			<td>Sigma-Aldrich</td>
			<td>B-1042</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Benzyl benzoate</td>
			<td>Sigma-Aldrich</td>
			<td>B-6630</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissecting microscope</td>
			<td>Leica</td>
			<td>M205A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Camera</td>
			<td>Leica</td>
			<td>DFC310FX</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ring light&#160;</td>
			<td>Leica&#160;</td>
			<td>MEB110</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Photoshop</td>
			<td>Adobe</td>
			<td>Photoshop 4.0</td>
		</tr>
	</tbody>
</table>

whole-mount imaging of mouse embryo sensory axon projections

胚における完全長の神経突起を可視化は、神経回路網の開発方法哺乳類の理解を得ることが不可欠である。ここでは、 その場でいくつかの遺伝子操作されたマウス系統を使用してそれらの表現型特性を評価するために、後根神経節（DRG）軸索突起のサブセットを標識するための方法を記載する。 TrkAの陽性ニューロンは、疼痛信号の送信専用の侵害受容ニューロンである。私たちは、そのままマウス胚内のすべてのTrkAの陽性末梢軸索の軌跡にラベルを付けるのTrkA taulacZマウスラインを利用しています。私たちは、さらに独立してニューロンの生存上の遺伝子操作の可能性のある影響の成長関連の質問を評価するために、本 ​​質的に神経細胞アポトーシスを廃止Baxのヌルバックグラウンド上にあるTrkA taulacZラインを繁殖。その後、関心の遺伝子改変マウスをTrkAのtaulと交配させるACZ / Baxの空行、その後、本明細書に記載の技術を用いた研究の準備ができている。このプレゼンテーションでは、マウスの繁殖計画の詳細については、解剖時のジェノタイピング、組織調製、染色および全体のマウント準備の完全長軸索軌道の可視化を可能にするためにクリアが含まれています。

TrkAのWT / taulacZの遺伝子型：たBax - / -およびTrkAのtaulacZ / taulacZ：たBax - / -胚は、明確に、標準的なPCR遺伝子型同定（ 図1）によって決定することができる。 X-gal染色ディスプレイは、従来、染色された胚（ 図2、図3a）に皮下末梢軸索アーバーを詳述し、そして組織のクリア後の胚全体（ 図3b、4）。 私...

Watch this Scientific Journal Video about Whole-mount Imaging of Mouse Embryo Sensory Axon Projections at JoVE.com

Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

We present here an optimized protocol to genotype, stain and prepare fetal mice for the imaging of peripheral nociceptor axon projections in the whole animal, as an effective method to assess sensory axon growth phenotypes in developing genetically engineered mice.

マウスの胚感覚軸索投影のホールマウントイメージング

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we ...

whole-mount-imaging-of-mouse-embryo-sensory-axon-projections

Research

JoVE Journal

Neuroscience

10.0K ビュー.  Weill Medical College of Cornell University. We present here an optimized protocol to genotype, stain and prepare fetal mice for the imaging of peripheral nociceptor axon projections in the whole animal, as an effective method to assess sensory axon growth phenotypes in developing genetically engineered mice.

ビデオ: Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

複数のマウスの神経解剖学的磁気共鳴イメージング

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

神経科学

Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and cones). The RPE is a monolayer of pigmented cuboidal cells and associates closely with the neural retina just above it. This association makes the RPE of great interest to researchers studying retinal diseases. The RPE is also the site of an in vivo assay of homology-directed DNA repair, the pun assay. The mouse eye is particularly difficult to dissect due to its small size (about 3.5mm in diameter) and its spherical shape. This article demonstrates in detail a procedure for dissection of the eye resulting in a whole mount of the RPE. In this procedure, we show how to work with, rather than against, the spherical structure of the eye. Briefly, the connective tissue, muscle, and optic nerve are removed from the back of the eye. Then, the cornea and lens are removed. Next, strategic cuts are made that result in significant flattening of the remaining tissue. Finally, the neural retina is gently lifted off, revealing an intact RPE, which is still attached to the underlying choroid and sclera. This whole mount can be used to perform the pun assay or for immunohistochemistry or immunofluorescent assessment of the RPE tissue.

A formal demonstration of the dissection of a mouse eye, resulting in a whole mount of the retinal pigment epithelium.

網膜色素上皮の全マウントするためのマウスの眼の解剖

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

ホールマウントマッスル分析による再現性のあるマウスの坐骨神経クラッシュと再生のその後の評価

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

Angiogenesis is the complex process of new blood vessel formation defined by the sprouting of new blood vessels from a pre-existing vessel network. Angiogenesis plays a key role not only in normal development of organs and tissues, but also in many diseases in which blood vessel formation is dysregulated, such as cancer, blindness and ischemic diseases. In adult life, blood vessels are generally quiescent so angiogenesis is an important target for novel drug development to try and regulate new vessel formation specifically in disease. In order to better understand angiogenesis and to develop appropriate strategies to regulate it, models are required that accurately reflect the different biological steps that are involved. The mouse neonatal retina provides an excellent model of angiogenesis because arteries, veins and capillaries develop to form a vascular plexus during the first week after birth. This model also has the advantage of having a two-dimensional (2D) structure making analysis straightforward compared with the complex 3D anatomy of other vascular networks. By analyzing the retinal vascular plexus at different times after birth, it is possible to observe the various stages of angiogenesis under the microscope. This article demonstrates a straightforward procedure for analyzing the vasculature of a mouse retina using fluorescent staining with isolectin and vascular specific antibodies.

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

The neonatal murine retina provides a well characterized physiological model of angiogenesis, which permits investigations of the roles of different genes or drugs that modulate angiogenesis in an in vivo context. Immunofluorescent staining to accurately visualize the vascular plexus is pivotal to the success of these types of studies.

血管新生を調査するために新生児マウス網膜の全体のマウント免疫蛍光染色<em生体内で&gt;</em

Angiogenesis  is the complex process of new blood vessel formation defined by the sprouting  of new blood vessels from a pre-existing vessel network. ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

における嗅覚受容ニューロンのホールマウント免疫標識<em&gt;ショウジョウバエ</em&gt;アンテナ

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Flat Mount Imaging of Mouse Skin and Its Application to the Analysis of Hair Follicle Patterning and Sensory Axon Morphology

Skin is a highly heterogeneous tissue. Intra-dermal structures include hair follicles, arrector pili muscles, epidermal specializations (such as Merkel cell clusters), sebaceous glands, nerves and nerve endings, and capillaries. The spatial arrangement of these structures is tightly controlled on a microscopic scale - as seen, for example, in the orderly arrangement of cell types within a single hair follicle - and on a macroscopic scale - as seen by the nearly identical orientations of thousands of hair follicles within a local region of skin. Visualizing these structures without physically sectioning the skin is possible because of the 2-dimensional geometry of this organ. In this protocol, we show that mouse skin can be dissected, fixed, permeabilized, stained, and clarified as an intact two dimensional object, a flat mount. The protocol allows for easy visualization of skin structures in their entirety through the full thickness of large areas of skin by optical sectioning and reconstruction. Images of these structures can also be integrated with information about position and orientation relative to the body axes.

Mammalian skin contains a diverse array of structures - such as hair follicles and nerve endings - that exhibit distinctive patterns of spatial organization. Analyzing skin as a flat mount takes advantage of the 2-dimensional geometry of this tissue to produce full-thickness high-resolution images of skin structures.

マウス皮膚や毛包パターニングおよび感覚軸索形態の解析への応用のフラットマウントイメージング

Skin is a highly heterogeneous tissue. Intra-dermal structures include hair follicles, arrector pili muscles, epidermal specializations (such as Merkel ...

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to blindness in many retinal diseases. Calcium (Ca2+), a key second messenger in photoreceptor signaling and metabolism, has been proposed to be indirectly linked with photoreceptor degeneration in various animal models. Systematically studying these aspects of cone physiology and pathophysiology has been hampered by the difficulties of electrically recording from these small cells, in particular in the mouse where the retina is dominated by rod photoreceptors. To circumvent this issue, we established a two-photon Ca2+ imaging protocol using a transgenic mouse line that expresses the genetically encoded Ca2+ biosensor TN-XL exclusively in cones and can be crossbred with mouse models for photoreceptor degeneration. The protocol described here involves preparing vertical sections (&#8220;slices&#8221;) of retinas from mice and optical imaging of light stimulus-evoked changes in cone Ca2+ level. The protocol also allows &#8220;in-slice measurement&#8221; of absolute Ca2+ concentrations; as the recordings can be followed by calibration. This protocol enables studies into functional cone properties and is expected to contribute to the understanding of cone Ca2+ signaling as well as the potential involvement of Ca2+ in photoreceptor death and retinal degeneration.

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

We describe a protocol to monitor Ca2+ dynamics in the axon terminals of cone photoreceptors using an ex-vivo&#160;slice preparation of the mouse retina. This protocol allows comprehensive studies of cone Ca2+ signaling in an important mammalian model system, the mouse.

イメージングカルシウム<sup&gt; 2+</supマウス網膜の光受容コーン軸索末端で&gt;ダイナミクス

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a spiral shape and have a tonotopic gradient for sound detection. The mouse cochlea is approximately 6 mm long and often divided into three turns (apex, middle, and base) for analysis. To investigate cell loss, cell division, or mosaic gene expression, the whole mount or surface preparation of the cochlea is useful. This dissection method allows visualization of all cells within the organ of Corti when combined with immunostaining and confocal microscopy to image cells at different planes in the z-axis. Multiple optical cross-sections can also be obtained from these z-stack images. In addition, the whole mount dissection method can be used for scanning electron microscopy, although a different fixation method is needed. Here, we present a method to isolate the organ of Corti as three intact cochlear turns (apex, middle, and base). This method can be used for mice ranging from one week of age through adulthood and differs from the technique used for neonatal samples where calcification of the cochlea is incomplete. A slightly modified version can be used for dissection of the rat cochlea. We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies.

We present a method to isolate the adult organ of Corti as three intact cochlear turns (apex, middle, and base). We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies. Together these techniques allow the study of hair cells, supporting cells, and other cell types found in the cochlea.

成体マウス蝸牛のホールマウント解剖および免疫蛍光

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a ...

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in vivo&#8212;rapidly, repetitively, and at a high resolution. This protocol describes OCT imaging in the mouse retina as a powerful tool to study optic neuropathies (OPN). The OCT system is an interferometry-based, non-invasive alternative to common post mortem histological assays. It provides a fast and accurate assessment of retinal thickness, allowing the possibility to track changes, such as retinal thinning or thickening. We present the imaging process and analysis with the example of the Opa1delTTAG mouse line. Three types of scans are proposed, with two quantification methods: standard and homemade calipers. The latter is best for use on the peripapillary retina during radial scans; being more precise, is preferable for analyzing thinner structures. All approaches described here are designed for retinal ganglion cells (RGC) but are easily adaptable to other cell populations. In conclusion, OCT is efficient in mouse model phenotyping and has the potential to be used for the reliable evaluation of therapeutic interventions.

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

This manuscript describes a protocol for the in vivo imaging of the mouse retina with high-resolution spectral domain optical coherence tomography (SD-OCT). It focuses on retinal ganglion cells (RGC) in the peripapillary region, with several scanning and quantifying approaches described.

光干渉断層計: マウス網膜神経節細胞In Vivoイメージング

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in ...

Quadruple Immunostaining of the Olfactory Bulb for Visualization of Olfactory Sensory Axon Molecular Identity Codes

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory Sensory Neuron (OSN) is a bipolar cell with a single dendrite and a single unbranched axon. An OSN expresses only one Olfactory Receptor (OR) gene, OSNs expressing a given type of OR converge their axons to a few sets of invariant glomeruli in the Olfactory Bulb (OB). A remarkable feature of OSN projection is that the expressed ORs play instructive roles in axonal projection. ORs regulate the expression of multiple axon-sorting molecules and generate the combinatorial molecular code of axon-sorting molecules at the OSN axon termini. Thus, to understand the molecular mechanisms of OR-specific axon guidance mechanisms, it is vital to characterize their expression profiles at the OSN axon termini within the same glomerulus. The aim of this article was to introduce methods for collecting as many glomeruli as possible on a single OB section and for performing immunostaining using multiple antibodies. This would allow the comparison and analysis of the expression patterns of axon-sorting molecules without staining variation between OB sections.

Olfactory sensory neurons express a wide variety of axon-sorting molecules to establish proper neural circuitry. This protocol describes an immunohistochemical staining method to visualize combinatorial expressions of axon-sorting molecules at the axon termini of olfactory sensory neurons.

嗅覚神経軸索分子識別コードの視覚化のための嗅球の四重免疫染色

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory ...