この研究は、NIHの助成金（R15DA029863）、フロリダアトランティック大学の学部研究助成金（M30014）と獣医学の研究助成金のロス大学によってサポートされていました。我々は、この作業のために（±）3,4-メチレンジ（±MDMA）を提供するための国立薬物乱用研究所（メリーランド州ロックビル）を感謝したいと思います。

以下で説明する動物の使用手順は、獣医、フロリダアトランティック大学のロス大学での施設内動物管理使用委員会（IACUC）によって承認されました。無菌テクニックと手袋が必要ですが、このプロトコルを使用している間、RNaseフリーの環境は必要ありません。 1.準備<ol><li>組織の調製と切片<ol><li>三つのグループに無作為にラットを割り当てます;、SAL /接頭辞および/ ​​3,4-メチレンジ接頭辞新鮮/生理食塩水（0.9％のNaCl SAL）（MDMAを、 表1で詳細を参照してください）。 </li><li> 2時間間隔でSALとMDMAのグループに、腹腔内に、それぞれ1ミリリットル/ kgのSALおよび10mg / kgのMDMAの3用量を投与。動物は6日間の治療を生き残るためにできるようにします。 </li><li> 7日目に、深い麻酔（ すなわち 、角膜の点滅の損失とTAのためにラットに腹腔内に5mg / kgのキシラジンの組み合わせでは100mg / kgのケタミンの投与IL）は反射神経をつまみます。 </li><li>新鮮/ SALグループの場合、速やかに（参考文献5で詳細を参照）、ラットを刎ねる、新鮮な脳のテストのための脳を削除します。 </li><li> SAL /接頭辞と接頭辞/ MDMAのグループの場合、胸骨約10 -12 cmのに沿ってカットを行い、その後、胸骨の終わりを公開します。 </li><li>止血剤と胸骨の端を持ち、鋭いハサミで両側に横方向に振動板をカットして、肋骨と肺に平行全体に上方カット。 </li><li>片手で、小さなピンセットで心臓の腹側先端を保持します。もう一方の手で、18 Gの注射針を用いて左心室を穿刺（針のアダプタ側を氷冷SAL及びパラホルムアルデヒドコンテナおよび蠕動ポンプにY字型のシリコンホースが接続されている注意してください。参照9を参照してください ）。 </li><li>流量（約5 ml /分）の中レベルのSALポンプの電源をオンにし、リターンCIRCの脱出を可能にするために鉗子で右心房を穿刺ピュレーション。 30分間SAL灌流を維持します。 </li><li>中レベルのパラホルムアルデヒドポンプの電源をオンにし、氷冷4％パラホルムアルデヒドを30分間動物を灌流することができます。 </li><li>灌流装置を取り外して、動物を首を切ります。頭蓋骨の正中線を切断posteroanteriorを行い、その後、頭蓋骨の空洞10を露出するために頭蓋骨フラップ。 </li><li>頭蓋骨の空洞からへらで脳を取り出し、4％パラホルムアルデヒドの25ミリリットルを含む50 ml遠心チューブに脳を置きます。 24時間4ºCの冷蔵庫でパラホルムアルデヒド含有チューブ内の脳をしてください。 </li><li> 3日間4ºCで30％ショ糖溶液25mlを含む50 mlの遠心管に脳に移動します。最後に、使用するまで-80℃でのビニール袋の中に脳を格納します。 </li><li>埋め込むメディアとチャックの脳をマウントし、-26ºCの温度でクライオスタットで脳を含む埋め込むメディアを凍結します。新鮮なフリーズをカットnは20μmのセクションに脳とパラホルムアルデヒド接頭辞脳10μmのセクションに。セクションにスライドに触れることにより、RTの顕微鏡スライドにセクションを転送します。 </li><li>空気乾燥、室温で4時間。使用するまで-80℃でタイトな密封された袋に切片スライドを保管してください。 </li></ol></li><li>脱水<ol><li> -80℃の冷凍庫からスライドを取り、3つのテストにセクションを割り当てる：HT2A、PPIB（ポジティブコントロール）またはのdapB（陰性対照）。ボールペンでマークとラベルのセクション（鉛筆は使用しないでください）​​。室温で5分間、100％アルコールでスライドを沈めます。ヒュームフード中で5分間スライドを乾燥させます。 </li></ol></li><li>前処理<ol><li>スライド上のセクションへの前処理-1試薬（アルカリホスファターゼの不活性化）のピペットを20μl。 （トレイが反応solutioからの蒸発を防止するための高湿度を維持するために、蓋で覆われていることに注意して水分トレイのラックレールにスライドを置きますN）。 </li><li>ゆっくり10分間低速で水平シェーカー上で水分トレイを振ります。 2分間の二重蒸留水 （ddH 2 O）で二回スライドを洗浄します。前処理-2試薬（パラホルムアルデヒド固定によって誘発される架橋結合の破壊）の150ミリリットルを含む200ミリリットルビーカーでスライドを沈めます。 100ºC（RNA構造11の熱による復元）で5分間の合計スライドをゆでます。 </li><li> 2分間のddH 2 Oで二回スライドを洗浄します。 5分間、100％アルコールでスライドを置きます。ヒュームフード中で5分間スライドを乾燥させます。組織セクションで選択した領域の周りに円を描くように疎水性のペンを使用して（例えば、 図2で説明したように視床と視床下部を含む非皮質構造）。 </li><li>ピペット10の各選択領域に前処理-3試薬（プローブの浸透の増加）のμL、および蓋を水分トレイをカバーしています。ハイブリダイゼーションオーブンでトレイを置きます水平シェーカーを搭載し、40ºCでオーブンの温度を設定します。 30分間穏やかトレイを振ります。 2分間のddH 2 Oで二回スライドを洗浄します。 </li></ol></li></ol> 2.ハイブリダイゼーションおよび増幅<ol><li>選択された領域にそれぞれHTR2A、PPIBとのdapBプローブ試薬のピペット10μL、。試薬の蒸発を防止するための蓋付き水分トレイを覆います。静かに低速で水平シェーカーセットに振ります。 2時間オーブンで40ºCでスライドをインキュベートします。 </li><li> 2分間の洗浄用緩衝液で2回スライドを洗浄します。選択した各領域に増幅-1試薬のピペット10μL、30分間40ºCでスライドを振るとインキュベートします。 </li><li> 2分間の洗浄用緩衝液で2回スライドを洗浄します。選択した各領域に増幅-2試薬ピペット10μL、15分間40ºCでスライドを振るとインキュベートします。 </li><li>スライドを2回洗浄ワット2分間の洗浄バッファー番目。選択した各領域に増幅-3試薬のピペット10μL、30分間40ºCでスライドを振るとインキュベートします。 </li><li> 2分間の洗浄用緩衝液で2回スライドを洗浄します。選択した各領域に増幅-4試薬のピペット10μL、15分間40ºCでスライドを振るとインキュベートします。 </li><li> 2分間の洗浄用緩衝液で2回スライドを洗浄します。選択した各領域に増幅-5試薬ピペット10μL、室温で30分間スライドを振るとインキュベートします。 </li><li> 2分間の洗浄用緩衝液で2回スライドを洗浄します。選択した各領域に増幅-6試薬ピペット10μL、15分間室温でスライドを振るとインキュベートします。 2分間の洗浄用緩衝液で2回スライドを洗浄します。 </li></ol> 3.信号検出<ol><li>選択した各エリアにレッド試薬のピペット10μL（試薬は1:60の比率で赤-BとRed-Aの混合物であると私使用されるべきであることに注意してくださいmmediately）。 RTで水平シェーカー上水分トレイにスライドを置き、10分間低速で軽く振ります。 </li><li> 10分間（のddH 2 O）で2回スライドを洗浄します。 15分間オーブンで60ºCでスライドを乾燥させます。 5分間ヒュームフードの下でキシレンにスライドを置きます。ピペット選択した各領域にキシレン系取り付けメディアの20μLとカバーガラスでカバーします。 </li></ol> 4.イメージキャプチャ<ol><li> 4×20×対物レンズの倍率で選択された領域（ 例えば、視床下部は、1と3図 ）のデジタル顕微鏡写真を取ります。画像ファイルを保存します。 </li></ol> 5.画像の粒子解析<ol><li>ドラッグして、メインImageJのウィンドウに画像ファイルをドロップします。メニューバーに移動し、「イメージ」を選択し、隣のドロップダウン・メニューから「タイプ」を選択し、「8ビット」を選択します。 </li><li>選択し、 &#39;のThrを選択し、「調整」esholdは &#39;ダイアログボックスを開きます。不要なバックグラウンドが除去されるように、ダイアログボックス内の下のバーの値を調整します。その後、2番目のダイアログボックスを開くには、「粒子の分析」を選択し、メニューバーに移動し、「分析」を選択します。 </li><li> 10で粒子のサイズを設定し、次の「表示のアウトライン」を選択し、「結果の表示」と「集計」ボックスをオンにします。最後に、ダイアログボックスでパーティクルデータを表示するには「OK」をクリックします。 </li></ol> 6.スプレッドシート計算<ol><li> Excelシートにデータを入力し、ベースラインとして、SALグループ内の粒子の数を平均します。各セクションのパーセンテージレベルを計算し、それに応じてグラフ（ 図4）を作ります。 </li></ol>

<ol>
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著者は、彼らが競合する金融利害関係を有していないことを宣言します。

One of the major concerns of in situ hybridization test is to choose appropriate techniques used for RNA preservation because of RNase enzymes. It is well known that these enzymes are widely present in the cells and environment which can affect the results. However, enzyme activity can be quickly distinguished by placing the tissue in the dry ice/alcohol solution5,12. Although quick preservation is critical for hybridization using a riboprobe13, little is known about experimental conditions...

Serotonin (5-hydroxytryptamine; 5-HT) syndrome is an acute neurologic disorder caused by 5-HT-promoting drugs such as antidepressants1, while also occurring in situations of MDMA use for recreational purposes2. Molecular mechanisms responsible for mood swings, learning and memory deficits that occur in association with the acute syndrome are not well understood3,4. In situ hybridization is a powerful research tool allowing the detection and quantification of specific mRNAs expressed potentially at a single-cell level. The conventional way to perform in situ hybridization is to utilize a radioactive-labeled riboprobe that specifically hybridizes the gene of interest. However, a major drawback is that the method requires complicated and time-consuming probe preparation and hybridization steps, as well as access to fresh frozen tissues maintained in an RNase-free environment5,6. 
Oligonucleotide probes have been recently developed to hybridize shorter RNA fragments than those required for riboprobes7. Furthermore, the probes produce a low background signal without sacrificing specificity8. This newly-developed probe technology can be used in situ hybridization on paraformaldehyde-prefixed brain tissues commonly available in immunocytochemical laboratories.
Here, we describe a protocol for in situ hybridization using oligonucleotide probes on paraformaldehyde-prefixed rat brain and compare findings with those noted in a fresh frozen brain5,6. This protocol is used to test the hypothesis that MDMA substance abuse causes changes in 5-HT2A receptor gene htr2a mRNA in the brain. We began the procedure with MDMA treatment followed by paraformaldehyde tissue perfusion of the animal, in situ hybridization of the thr2a probe, and data analysis. Note that dapB (the bacterial gene coding for dihydrodipicolinate reductase) is used as a negative control, and ppiB (housekeeping gene coding for peptidylprolyl isomerase B) as a positive control.

<table border="0" cellpadding="0" cellspacing="0" width="873">
	<tbody>
		<tr>
			<td>RNAscope Negative Control Probe-DapB</td>
			<td>Advanced cell diagnostic, INC</td>
			<td>310098</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAscope Pretreat 4</td>
			<td>Advanced cell diagnostic, INC</td>
			<td>320046</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAscope 2.0 HD Reagent Kit - Red</td>
			<td>Advanced cell diagnostic, INC</td>
			<td>310036</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAscope Probe - Rn-Ppib</td>
			<td>Advanced cell diagnostic, INC</td>
			<td>313921</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat Htr2a</td>
			<td>Advanced cell diagnostic, INC</td>
			<td>300031</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Leica</td>
			<td>CM 1850</td>
			<td></td>
		</tr>
		<tr>
			<td>Horizontal shaker</td>
			<td>VWR</td>
			<td>88032-088</td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridization oven</td>
			<td>Thermo Fisher Scientific</td>
			<td>222000</td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost Plus microscope slide</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrophobic pen</td>
			<td>Vector</td>
			<td>H-4000</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus</td>
			<td>Provis AX70</td>
			<td></td>
		</tr>
	</tbody>
</table>

rapid in situ hybridization using oligonucleotide probes on paraformaldehyde-prefixed brain of rats with serotonin syndrome

3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) toxicity may cause region-specific changes in serotonergic mRNA expression due to acute serotonin (5-hydroxytryptamine; 5-HT) syndrome. This hypothesis can be tested using in situ hybridization to detect the serotonin 5-HT2A receptor gene htr2a. In the past, such procedures, utilizing radioactive riboprobe, were difficult because of the complicated workflow that needs several days to perform and the added difficulty that the technique required the use of fresh frozen tissues maintained in an RNase-free environment. Recently, the development of short oligonucleotide probes has simplified in situ hybridization procedures and allowed the use of paraformaldehyde-prefixed brain sections, which are more widely available in laboratories. Here, we describe a detailed protocol using non-radioactive oligonucleotide probes on the prefixed brain tissues. Hybridization probes used for this study include dapB (a bacterial gene coding for dihydrodipicolinate reductase), ppiB (a housekeeping gene coding for peptidylprolyl isomerase B), and htr2a (a serotonin gene coding for 5-HT2A receptors). This method is relatively simply, cheap, reproducible and requires less than two days to complete.

（NT &lt;25）オリゴヌクレオチドのRNAプローブを用いて、ハイブリダイゼーションはパラホルムアルデヒド、接頭辞及び新鮮凍結脳から調製した視床下部細胞における赤い点として検出することができます。 HTR2Aの mRNA分子は、（実線の矢印で示される）いくつかの細胞中に存在するが、他のものではない（白抜き矢印）。我々は、パラホルムアルデヒド、接頭辞及び新鮮凍結組織（ <st...

Watch this Scientific Journal Video about Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome at JoVE.com

Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

This protocol describes a rapid and simplified in situ hybridization method ideal forparaformaldehyde-prefixed brain, thus reducing the need for prolonged complex steps while using fresh frozen tissues. The method is validated using the identification of the serotonin 5-HT2A receptor gene htr2a in rats.

ラピッド<em&gt;その場で</emセロトニン症候群のラットのパラホルムアルデヒド接頭辞脳にオリゴヌクレオチドプローブを使用して&gt;ハイブリダイゼーション

3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) toxicity may cause region-specific changes in serotonergic mRNA expression due to acute serotonin ...

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Research

JoVE Journal

Neuroscience

Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

8.8K ビュー.  Ross University School of Veterinary Medicine. This protocol describes a rapid and simplified in situ hybridization method ideal forparaformaldehyde-prefixed brain, thus reducing the need for prolonged complex steps while using fresh frozen tissues. The method is validated using the identification of the serotonin 5-HT2A receptor gene htr2a in rats.

ビデオ: Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

Double Fluorescence in situ Hybridization in Fresh Brain Sections

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh frozen brain sections. Our group has successfully used this approach to study gene co-regulation. More specifically, we have used this dFISH method to explore the anatomical organization, neurochemical properties, and the impact of sensory experience in central sensory circuits, at single cell resolution. This protocol has been validated in brain tissue from mice, rats and songbirds but is expected to be easily adaptable to other vertebrate species, as well as to an array of non-neural tissues. In this film we provide a detailed demonstration of the main steps of this procedure.

Double Fluorescence in situ Hybridization in Fresh Brain Sections

This protocol involves a non-radioactive in-situ hybridization procedure that enables the simultaneous identification of two transcript species, at a single cell resolution, in thin sections of the vertebrate brain.

二重蛍光その場で</em新鮮な脳切片における>ハイブリダイゼーション

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh ...

神経科学

TMS: Using the Theta-Burst Protocol to Explore Mechanism of Plasticity in Individuals with Fragile X Syndrome and Autism

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS display abnormalities in the expression of FMR1 - a protein required for typical, healthy neural development.3 Recent data has suggested that the loss of this protein can cause the cortex to be hyperexcitable thereby affecting overall patterns of neural plasticity.4,5
In addition, Fragile X shows a strong comorbidity with autism: in fact, 30% of children with FXS are diagnosed with autism, and 2 - 5% of autistic children suffer from FXS.6
Transcranial Magnetic Stimulation (a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability via the application of localized magnetic field pulses 7,8) represents a unique method of exploring plasticity and the manifestations of FXS within affected individuals. More specifically, Theta-Burst Stimulation (TBS), a specific stimulatory protocol shown to modulate cortical plasticity for a duration up to 30 minutes after stimulation cessation in healthy populations, has already proven an efficacious tool in the exploration of abnormal plasticity.9,10 
Recent studies have shown the effects of TBS last considerably longer in individuals on the autistic spectrum - up to 90 minutes.11 This extended effect-duration suggests an underlying abnormality in the brain's natural plasticity state in autistic individuals - similar to the hyperexcitability induced by Fragile X Syndrome.
In this experiment, utilizing single-pulse motor-evoked potentials (MEPs) as our benchmark, we will explore the effects of both intermittent and continuous TBS on cortical plasticity in individuals suffering from FXS and individuals on the Autistic Spectrum.

In this article, we examine the effects of Theta-Burst TMS stimulation on cortical plasticity in individuals suffering from Fragile X syndrome and individuals on the autistic spectrum.

TMS：脆弱X症候群と自閉症の可塑性のMechasnismを探検するシータバーストプロトコルを使用する

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS ...

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

Going beyond single gene function to cut deeper into gene regulatory networks requires multiple mutations combined in a single animal. Such analysis of two or more genes needs to be complemented with in situ hybridization of other genes, or immunohistochemistry of their proteins, both in whole mounted developing organs or sections for detailed resolution of the cellular and tissue expression alterations. Combining multiple gene alterations requires the use of cre or flipase to conditionally delete genes and avoid embryonic lethality. Required breeding schemes dramatically enhance effort and cost proportional to the number of genes mutated, with an outcome of very few animals with the full repertoire of genetic modifications desired. Amortizing the vast amount of effort and time to obtain these few precious specimens that are carrying multiple mutations necessitates tissue optimization. Moreover, investigating a single animal with multiple techniques makes it easier to correlate gene deletion defects with expression profiles. We have developed a technique to obtain a more thorough analysis of a given animal; with the ability to analyze several different histologically recognizable structures as well as gene and protein expression all from the same specimen in both whole mounted organs and sections. Although mice have been utilized to demonstrate the effectiveness of this technique it can be applied to a wide array of animals. To do this we combine lipophilic dye tracing, whole mount in situ hybridization, immunohistochemistry, and histology to extract the maximal possible amount of data.

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

A combination of different techniques to maximize data collection from mouse tissue is presented.

親油性色素を組み合わせることで、その場でハイブリダイゼーション、免疫組織化学、および組織学

Going beyond single gene function to cut deeper into gene regulatory networks requires multiple mutations combined in a single animal.  Such analysis of ...

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several weeks, and this is accompanied by short distance regeneration (a few mm) of propriospinal axons and spinal-projecting axons from the brainstem. Among the 36 large identifiable spinal-projecting neurons, some are good regenerators and others are bad regenerators. These neurons can most easily be identified in wholemount CNS preparations. In order to understand the neuron-intrinsic mechanisms that favor or inhibit axon regeneration after injury in the vertebrates CNS, we determine differences in gene expression between the good and bad regenerators, and how expression is influenced by spinal cord transection. This paper illustrates the techniques for housing larval and recently transformed adult sea lampreys in fresh water tanks, producing complete spinal cord transections under microscopic vision, and preparing brain and spinal cord wholemounts for in situ hybridization. Briefly, animals are kept at 16&#160;&#176;C and anesthetized in 1% Benzocaine in lamprey Ringer. The spinal cord is transected with iridectomy scissors via a dorsal approach and the animal is allowed to recover in fresh water tanks at 23 &#176;C. For in situ hybridization, animals are reanesthetized and the brain and cord removed via a dorsal approach.

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

Lampreys recover locomotion after a complete spinal cord injury. However, some spinal-projecting neurons are good regenerators and others are not. This paper illustrates the techniques for housing sea lamprey larvae (and recently transformed adults), producing complete spinal cord transections and preparing wholemount brains and spinal cords for in situ hybridization.

その後のホールマウントのための完全な脊髄損傷や脳解剖プロトコル<em&gt;その場で</em&gt;幼虫海ヤツメウナギにおけるハイブリダイゼーション

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several ...

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for maintenance, repair or neurodegeneration in postdevelopmental periods. High throughput analyses have recently revealed that axons have a more dynamic and complex transcriptome than previously expected. These analysis, however have been mostly done in cultured neurons where axons can be isolated from the somato-dendritic compartments. It is virtually impossible to achieve such isolation in whole tissues in vivo. Thus, in order to verify the recruitment of mRNAs and their functional relevance in a whole animal, transcriptome analyses should ideally be combined with techniques that allow the visualization of mRNAs in situ. Recently, novel ISH technologies that detect RNAs at a single-molecule level have been developed. This is especially important when analyzing the subcellular localization of mRNA, since localized RNAs are typically found at low levels. Here we describe two protocols for the detection of axonally-localized mRNAs using a novel ultrasensitive RNA ISH technology. We have combined RNAscope ISH with axonal counterstain using fluorescence immunohistochemistry or histological dyes to verify the recruitment of Atf4 mRNA to axons in vivo in the mature mouse and human brains.

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

RNA in situ hybridization (ISH) enables the visualization of RNAs in cells and tissues. Here we show how combination of RNAscope ISH with immunohistochemistry or histological dyes can be successfully used to detect mRNAs localized to axons in sections of mouse and human brains.

高解像度を使用した脳切片における軸索のローカライズされたmRNAの検出<em&gt;その場での</em&gt;ハイブリダイゼーション

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for ...

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

Detection of gene expression in different types of brain cells e.g., neurons, astrocytes, oligodendrocytes, oligodendrocyte precursors and microglia, can be hampered by the lack of specific primary or secondary antibodies for immunostaining. Here we describe a protocol to detect the expression of three different genes in the same brain section using double fluorescence in situ hybridization with two gene-specific probes followed by immunostaining with an antibody of high specificity directed against the protein encoded by a third gene. The Aspartoacyclase (ASPA) gene, mutations of which can lead to a rare human white matter disease - Canavan disease - is thought to be expressed in oligodendrocytes and microglia but not in astrocytes and neurons. However, the precise expression pattern of ASPA in the brain has yet to be established. This protocol has allowed us to determine that ASPA is expressed in a subset of mature oligodendrocytes and it can be generally applied to a wide range of gene expression pattern studies.

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

Localizing gene expression to specific cell types can be challenging due to the lack of specific antibodies. Here we describe a protocol for simultaneous triple detection of gene expression by combining double fluorescence RNA in situ hybridization with immunostaining.

ダブル蛍光を組み合わせます<em&gt;その場で</em&gt;マウス脳切片の3遺伝子の発現を検出するための免疫標識とのハイブリダイゼーション

Detection of gene expression in different types of brain cells e.g., neurons, astrocytes, oligodendrocytes, oligodendrocyte precursors and microglia, can ...

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory Receptor Neurons (ORNs) housed in hair-like structures, called chemosensilla, of the antennae. On the ORNs&#39; membranes, Odorant Receptors (ORs) are believed to be involved in odor coding. Thus, being able to identify genes localized to the ORNs is necessary to recognize OR genes, and provides a fundamental basis for further functional in situ studies. The RNA expression levels of specific ORs in insect antennae are very low, and preserving insect tissue for histology is challenging. Thus, it is difficult to localize an OR to a specific type of sensilla using RNA in situ hybridization. In this paper, a detailed and highly effective RNA in situ hybridization protocol particularly for lowly expressed OR genes of insects, is introduced. In addition, a specific OR gene was identified by conducting double-color fluorescent in situ hybridization experiments using a co-expressing receptor gene, Orco, as a marker.

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

This paper describes a detailed and highly effective RNA in situ hybridization protocol particularly for low-level expressed Odorant Receptor (OR) genes, as well as other genes, in insect antennae using digoxigenin (DIG)-labeled or biotin-labeled probes.

Locust AntennaeにおけるRNAによる嗅覚受容体遺伝子の局在<em&gt;現場で</em&gt;ハイブリダイゼーション

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory ...

Two-photon Imaging of Microglial Processes' Attraction Toward ATP or Serotonin in Acute Brain Slices

Microglial cells are resident innate immune cells of the brain that constantly scan their environment with their long processes and, upon disruption of homeostasis, undergo rapid morphological changes. For example, a laser lesion induces in a few minutes an oriented growth of microglial processes, also called &#34;directional motility&#34;, toward the site of injury. A similar effect can be obtained by delivering locally ATP or serotonin (5-hydroxytryptamine [5-HT]). In this article, we describe a protocol to induce a directional growth of microglial processes toward a local application of ATP or 5-HT in acute brain slices of young and adult mice and to image this attraction over time by multiphoton microscopy. A simple method of quantification with free and open-source image analysis software is proposed. A challenge that still characterizes acute brain slices is the limited time, decreasing with age, during which the cells remain in a physiological state. This protocol, thus, highlights some technical improvements (medium, air-liquid interface chamber, imaging chamber with a double perfusion) aimed at optimizing the viability of microglial cells over several hours, especially in slices from adult mice.

Two-photon Imaging of Microglial Processes&#039; Attraction Toward ATP or Serotonin in Acute Brain Slices

Microglia, the resident immune cells of the brain, respond quickly with morphological changes to modifications of their environment. This protocol describes how to use two-photon microscopy to study the attraction of microglial processes toward serotonin or ATP in acute brain slices of mice.

ATP または急性脳スライスにおけるセロトニンに対するミクログリアのプロセスの魅力の二光子励起イメージング

Microglial cells are resident innate immune cells of the brain that constantly scan their environment with their long processes and, upon disruption of ...

Induction of Diffuse Axonal Brain Injury in Rats Based on Rotational Acceleration

Traumatic brain injury (TBI) is a major cause of death and disability. Diffuse axonal injury (DAI) is the predominant mechanism of injury in a large percentage of TBI patients requiring hospitalization. DAI involves widespread axonal damage from shaking, rotation or blast injury, leading to rapid axonal stretch injury and secondary axonal changes that are associated with a long-lasting impact on functional recovery. Historically, experimental models of DAI without focal injury have been difficult to design. Here we validate a simple, reproducible and reliable rodent model of DAI that causes widespread white matter damage without skull fractures or contusions.

This protocol validates a reliable, easy-to-perform and reproducible rodent model of brain diffuse axonal injury (DAI) that induces widespread white matter damage without skull fractures or contusions.

回転加速度に基づくラットにおけるびまん軸索脳損傷の誘導

Traumatic brain injury (TBI) is a major cause of death and disability. Diffuse axonal injury (DAI) is the predominant mechanism of injury in a large ...

Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections

Fluorescent in situ hybridization (FISH) is a molecular technique that identifies the presence and spatial distribution of specific RNA transcripts within cells. Neurochemical phenotyping of functionally identified neurons usually requires concurrent labelling with multiple antibodies (targeting protein) using immunohistochemistry (IHC) and optimization of in situ hybridization (targeting RNA), in tandem. A &#34;neurochemical signature&#34; to characterize particular neurons may be achieved however complicating factors include the need to verify FISH and IHC targets before combining the methods, and the limited number of RNAs and proteins that may be targeted simultaneously within the same tissue section.
Here we describe a protocol, using both fresh frozen and fixed mouse brain preparations, which detects multiple mRNAs and proteins in the same brain section using RNAscope FISH followed by fluorescence immunostaining, respectively. We use the combined method to describe the expression pattern of low abundance mRNAs (e.g., galanin receptor 1) and high abundance mRNAs (e.g., glycine transporter 2), in immunohistochemically identified brainstem nuclei.
Key considerations for protein labelling downstream of the FISH assay extend beyond tissue preparation and optimization of FISH probe labelling. For example, we found that antibody binding and labelling specificity can be detrimentally affected by the protease step within the FISH probe assay. Proteases catalyze hydrolytic cleavage of peptide bonds, facilitating FISH probe entry into cells, however they may also digest the protein targeted by the subsequent IHC assay, producing off target binding. The subcellular location of the targeted protein is another factor contributing to IHC success following FISH probe assay. We observed IHC specificity to be retained when the targeted protein is membrane bound, whereas IHC targeting cytoplasmic protein required extensive troubleshooting. Finally, we found handling of slide-mounted fixed frozen tissue more challenging than fresh frozen tissue, however IHC quality was overall better with fixed frozen tissue, when combined with RNAscope.

Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections

This protocol describes a method for combining fluorescence in situ hybridization (FISH) and fluorescence immunohistochemistry (IHC) in both fresh frozen and fixed mouse brain sections, with the goal of achieving multilabel FISH and fluorescence IHC signal. IHC targeted cytoplasmic and membrane attached proteins.

マルチプレックス蛍光 In Situ ハイブリダイゼーションと蛍光免疫組織化学の融合による新鮮、凍結、または固定マウスの脳切片の結合(英語)

Fluorescent in situ hybridization (FISH) is a molecular technique that identifies the presence and spatial distribution of specific RNA transcripts within ...