We thank the Advanced Microscopy Unit (School of Life Sciences, University of Nottingham) and the Histology team of MRC Human Reproductive Sciences Unit (Edinburgh), the Institute of Neuroscience at the ULB and the FNRS for help and support. This work was supported by MRC (MR/L001047/1 to A.D.J.).

全ての動物-関与手順はノッティンガムの倫理審査委員会の大学に従って行われました。 1.免疫染色のための適切な組織標本を選択します<ol><li>以前25記載されているように、野生型CD1マウス胚および成体の脳組織のパラフィン包埋切片を生成します。方法25の免疫染色のために4％ホルムアルデヒド（FA）または4％パラホルムアルデヒド（PFA）のいずれかで固定した脳組織切片を使用します。 注：パラフィン包埋組織切片の使用は、抗体標識の前に脱脂が必要です。 2を用いた治療ので - DNA変性のためのプロトコルで用いられる4 M塩酸（HCl）、最も抗原回復戦略とは互換性がありません、我々は、タンパク質と5MC酸化誘導体の同時検出のために冷凍のかミクロトームセクションのいずれかを使用することをお勧めしますマーカー。 </li></ol> 2.脱ロウパラフィン包埋組織切片&lt;<html> / P&gt; <ol><li>実行中のクラスII安全キャビネット内で、室温で、キシレンで満たされたコプリンジャー中で10分間2回ずつパラフィン包埋組織切片を洗浄します。 注：これは、不完全なパラフィン除去が一貫性のない染色パターンにつながることができますように新鮮なキシレンを使用することが重要です。 </li><li>脱ワックス後、急速に95、75、そして室温で10分間、50％エタノール中で連続して洗浄す​​ることにより組織切片を再水和します。 </li></ol> 3.固定および冷凍のとミクロトーム切片の透過処理<ol><li> RTで15分間、氷冷4％PFAまたは4％FAのいずれかでそれらを置くことによって再水和した組織切片を修正しました。 </li><li>室温で5分間、PBS中のセクション（リン酸緩衝生理食塩水）を洗浄することにより、過剰な固定液を除去します。 </li><li> RTで30分間PBXが充填されたコプリンジャー（PBS中0.5％トリトンX-100）中に配置することによって、組織切片を透過。 PBT（PBS中0.01％のTween 20）でまもなくセクションを洗浄することにより、過剰なPBXを削除してください。 </li></ol>jove_title &quot;&gt; 4。オキシナイト5MC誘導体の免疫染色<ol><li> DNAの脱プリンをRTで60分間、2 N HCl中で透過処理セクションを配置します。 注：より効率的なDNA変性へのHCl（ 例えば、4 N）の鉛のより高い濃度が、これらはDAPIでオキシ-MCSの同時検出のために互換性がありません。 </li><li>塩酸を中和するために、室温で30分間、10mMトリス - 塩酸（pH8.5）中のセクションを配置します。また、PBS中で5分ごとにセクションを3回洗浄します。室温で5分間PBTのセクションをインキュベートします。 </li><li>慎重に組織切片を静かにスライドを振盪することにより、任意のステップで完全に乾燥させることなく、組織切片を周囲から液体を除去します。セクションに触れずにセクションを取り囲むように、疎水性バリアペンを使用してください。 注：疎水性バリアペンは、組織を染色するために必要な抗体の量を減少させ、複数のセクションがdifferenで染色することができるようにすることができます同じスライド上のトン抗体。 </li><li>湿潤チャンバー中、室温で1時間ブロッキング溶液（PBS中10％のウシ血清アルブミン）100μl中のセクションをインキュベートします。 注：このステップをスキップする修正されたシトシン塩基を染色の効率に影響を及ぼさないであろう。 </li><li>マウスモノクローナル抗5hmC 5,000希釈液と1：湿潤チャンバー内で室温で1時間、ブロッキング溶液中で、ウサギポリクローナル抗5caC一次抗体を1000倍希釈液100μlで組織切片をインキュベートします。必要に応じて別の方法として、4℃で一晩のインキュベーションを行います。 注：一次抗体なしで処理セクションは、染色手順のためのような適切な陰性対照を提供することができます。 5caC、5FCと5hmC 20に富むポスト交尾マウス胚性脳切片を陽性対照として使用することができます12.5日。 </li><li>コップで5分間、PBTで満たされたコプリンジャーに各3回セクションを洗浄することにより過剰な抗体を除去RTで林の瓶。 注：洗浄溶液の体積を増加させるバックグラウンド染色を減少させることができます。 </li><li>過剰PBTを削除して、必要に応じて、PBTは、疎水性の障壁を弱めることができ、界面活性剤が含まれているとして、疎水性のバリアペンで再びセクションを取り囲みます。 </li><li>ヤギ抗ウサギHRP結合抗体の400希釈および1：ブロッキング溶液中のロバ抗マウス555結合抗体の400倍希釈してください。 </li><li>湿潤チャンバー中、室温で1時間、 ステップ4.9からの二次抗体混合物100μl中の組織切片をインキュベートします。 </li><li>室温で5分間ずつPBT 3回満たしコプリンジャーに組織切片を洗浄してください。 </li><li>室温で2分間、チラミドシグナル増幅バッファ内のチラミドの200希釈：1100μlの組織切片を置きます。 </li><li> すぐに、PBTで5分間スライドをそれぞれ3回洗浄することによって過剰チラミド溶液を除去。 </li><li>気をつけyが過剰PBTを削除し、 すぐに （材料リストを参照してください）封入剤のドロップを持つセクションをカバーしています。 </li><li>静かに組織切片上のカバースリップを置き、 すぐにマニキュアでカバーガラスをシール。 </li><li>顕微鏡検査の前に、いくつかの時間のために4℃で組織切片を保管してください。 （ - 40X倍率10）405、488および/または555 nmで蛍光顕微鏡下で調べます。 </li></ol>

<ol>
	<li>Bird, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+methylation+patterns+and+epigenetic+memory.">DNA methylation patterns and epigenetic memory.</a> Genes Dev. 16, 6-21 (2002).</li><li>Reik, W., Dean, W., Walter, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+reprogramming+in+mammalian+development.">Epigenetic reprogramming in mammalian development.</a> Science. 293, 1089-1093 (2001).</li><li>Jaenisch, R., Bird, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+regulation+of+gene+expression:+how+the+genome+integrates+intrinsic+and+environmental+signals.">Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals.</a> Nat Genet. 33, 245-254 (2003).</li><li>Li, E., Bestor, T. H., Jaenisch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+mutation+of+the+DNA+methyltransferase+gene+results+in+embryonic+lethality.">Targeted mutation of the DNA methyltransferase gene results in embryonic lethality.</a> Cell. 69 (6), 915-926 (1992).</li><li>Okano, 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=DNA+methyltransferases+Dnmt3a+and+Dnmt3b+are+essential+for+de+novo+methylation+and+mammalian+development.">DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development.</a> Cell. 99 (3), 247-257 (1999).</li><li>Cedar, H., Bergman, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Programming+of+DNA+methylation+patterns.">Programming of DNA methylation patterns.</a> Annu Rev Biochem. 81, 97-117 (2012).</li><li>Amir, R. E., 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=Rett+syndrome+is+caused+by+mutations+in+X-linked+MECP2,+encoding+methyl-CpG-binding+protein+2.">Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2.</a> Nat Genet. 23, 185-188 (1999).</li><li>Chouliaras, 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=Consistent+decrease+in+global+DNA+methylation+and+hydroxymethylation+in+the+hippocampus+of+Alzheimer's+disease+patients.">Consistent decrease in global DNA methylation and hydroxymethylation in the hippocampus of Alzheimer's disease patients.</a> Neurobiol Aging. 34, 2091-2099 (2013).</li><li>Jowaed, 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=Methylation+regulates+alpha-synuclein+expression+and+is+decreased+in+Parkinson's+disease+patients'+brains.">Methylation regulates alpha-synuclein expression and is decreased in Parkinson's disease patients' brains.</a> J Neurosci. 30, 6355-6359 (2010).</li><li>Reik, W., 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=Age+at+onset+in+Huntington's+disease+and+methylation+at+D4S95.">Age at onset in Huntington's disease and methylation at D4S95.</a> J Med Genet. 30, 185-188 (1993).</li><li>Landgrave-G&#242;mez, J., Mercado-G&#242;mez, O., Guevara-Guzm&#225;n, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+mechanisms+in+neurological+and+neurodegenerative+diseases.">Epigenetic mechanisms in neurological and neurodegenerative diseases.</a> Front Cell Neurosci. 27, 9-58 (2015).</li><li>Seisenberger, S., Peat, J. R., Hore, T. A., Santos, F., Dean, W., Reik, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+DNA+methylation+in+the+mammalian+life+cycle:+building+and+breaking+epigenetic+barriers.">Reprogramming DNA methylation in the mammalian life cycle: building and breaking epigenetic barriers.</a> Philos Trans R Soc Lond B Biol Sci. 368 (1609), (2013).</li><li>Wu, H., Zhang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+and+functions+of+Tet+protein-mediated+5-+methylcytosine+oxidation.">Mechanisms and functions of Tet protein-mediated 5- methylcytosine oxidation.</a> Genes Dev. 25, 2436-2452 (2011).</li><li>Oswald, J., 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=Active+demethylation+of+the+paternal+genome+in+the+mouse+zygote.">Active demethylation of the paternal genome in the mouse zygote.</a> Curr Biol. 10 (8), 475-478 (2000).</li><li>Ooi, S. K., Bestor, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+colorful+history+of+active+DNA+demethylation.">The colorful history of active DNA demethylation.</a> Cell. 133 (7), 1145-1148 (2008).</li><li>Kriaucionis, S., Heintz, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nuclear+DNA+base+5-hydroxymethylcytosine+is+present+in+Purkinje+neurons+and+the+brain.">The nuclear DNA base 5-hydroxymethylcytosine is present in Purkinje neurons and the brain.</a> Science. 324, 929-930 (2009).</li><li>Tahiliani, 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=Conversion+of+5-methylcytosine+to+5-hydroxymethylcytosine+in+mammalian+DNA+by+MLL+partner+TET1.">Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1.</a> Science. 324, 930-935 (2009).</li><li>Ito, S., 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=Tet+proteins+can+convert+5-methylcytosine+to+5-formylcytosine+and+5-carboxylcytosine.">Tet proteins can convert 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine.</a> Science. 333, 1300-1303 (2011).</li><li>He, Y. F., 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=Tet-mediated+formation+of+5-carboxylcytosine+and+its+excision+by+TDG+in+mammalian+DNA.">Tet-mediated formation of 5-carboxylcytosine and its excision by TDG in mammalian DNA.</a> Science. 333, 1303-1307 (2011).</li><li>Wheldon, L. 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=Transient+accumulation+of+5-carboxylcytosine+indicates+involvement+of+active+demethylation+in+lineage+specification+of+neural+stem+cells.">Transient accumulation of 5-carboxylcytosine indicates involvement of active demethylation in lineage specification of neural stem cells.</a> Cell Rep. 7, 1353-1361 (2014).</li><li>Yu, 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=Base-resolution+analysis+of+5-hydroxymethylcytosine+in+the+mammalian+genome.">Base-resolution analysis of 5-hydroxymethylcytosine in the mammalian genome.</a> Cell. 149, 1368-1380 (2012).</li><li>Lu, X., 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=Chemical+modification+assisted+bisulfite+sequencing+(CAB-Seq)+for+5-carboxylcytosine+detection+in+DNA.">Chemical modification assisted bisulfite sequencing (CAB-Seq) for 5-carboxylcytosine detection in DNA.</a> J Am Chem Soc. 26, 9315-9317 (2013).</li><li>Globisch, D., 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=Tissue+distribution+of+5-hydroxymethylcytosine+and+search+for+active+demethylation+intermediates.">Tissue distribution of 5-hydroxymethylcytosine and search for active demethylation intermediates.</a> PLoS One. 5, e15367 (2010).</li><li>Szwagierczak, 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=Sensitive+enzymatic+quantifi+cation+of+5-+hydroxymethylcytosine+in+genomic+DNA.">Sensitive enzymatic quantifi cation of 5- hydroxymethylcytosine in genomic DNA.</a> Nucleic Acids Res. 38, e181 (2010).</li><li>Ruzov, 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=Lineage-specific+distribution+of+high+levels+of+genomic+5-hydroxymethylcytosine+in+mammalian+development.">Lineage-specific distribution of high levels of genomic 5-hydroxymethylcytosine in mammalian development.</a> Cell Res. 21, 1332-1334 (2011).</li><li>Almeida, R. D., 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=Semi-quantitative+immunohistochemical+detection+of+5-hydroxymethyl-cytosine+reveals+conservation+of+its+tissue+distribution+between+amphibians+and+mammals.">Semi-quantitative immunohistochemical detection of 5-hydroxymethyl-cytosine reveals conservation of its tissue distribution between amphibians and mammals.</a> Epigenetics. 7, 137-140 (2012).</li><li>Lister, R., 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=Global+epigenomic+reconfiguration+during+mammalian+brain+development.">Global epigenomic reconfiguration during mammalian brain development.</a> Science. 341, 1237905 (2013).</li><li>Santos, F., Dean, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+11,+Using+immunofluorescence+to+observe+methylation+changes+in+mammalian+preimplantation+embryos.">Chapter 11, Using immunofluorescence to observe methylation changes in mammalian preimplantation embryos.</a> Nuclear reprogramming methods and Protocols. , 129-138 (2006).</li><li>Wang, 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=Molecular+basis+for+5-carboxycytosine+recognition+by+RNA+polymerase+II+elongation+complex.">Molecular basis for 5-carboxycytosine recognition by RNA polymerase II elongation complex.</a> Nature. 523, 621-625 (2015).</li></ol>

The authors declare no conflict of interest.

いくつかの組織で5MC酸化誘導体、5FCと5caCの報告低量は、標準的な免疫化学プロトコルのために重大な制限を提示するであろうが、ペルオキシダーゼ結合二次抗体の取り込みは、固定組織や細胞におけるこれらのシトシン修飾の検出を可能にした（ 図2） 。しかし、チラミド溶液と最適なインキュベーション時間は、信号強度とチラミドベースの信号増幅の期間の間に直線関係が詳細については、観察されたチラミドシグナル増幅キットの各個々のバッチについて実験的に最適化する必要がありアルメイダらを参照してください。2012年26 。さらに、信号/背景比が著しく過剰な抗体の効率的な除去を可能にするためにコプリンジャー中での洗浄を実施することによって向上させることができます。チラミド溶液とのインキュベーション後、直ちにDにPBT溶液中で洗浄することにより反応を停止することが重要ですecreaseバックグラウンド染色。セクションでは、手順の間の任意の時点で乾燥できるようにするために重要ではありません。 DNAの脱プリン化の効率は、37℃で脱プリン反応を行うことにより改善することができます。それは、二本鎖DNAと排他的に相互作用する4 N HClを使用しての代わりに2ながらNは、DAPIで共染色することを認めていないDNAの脱プリン化のために4 N HClを用いて5MCの修飾された形態の染色を強化します。 この技法は、検出可能な、それは5MCまたはその酸化誘導体の絶対的なレベルを評価するために使用することができないシトシン塩基の修飾された形態の強固な半定量的評価を提供することができません。したがって、我々は他の補完の使用をお勧めしますが、量的には16、19 -24に近づきます。哺乳類のゲノム中の5MC酸化誘導体の発見以来、いくつかのアプローチは、それらの生物学的役割16、19-24を研究するために開発されてきました。これらapproaが、CHESは5MC酸化誘導体の絶対レベルを決定する上で貴重なことができ、彼らは彼らの空間分布20、26に関する情報を提供していません。我々は成功し、異なる細胞型で5MC酸化誘導体の空間分布及び局在をマッピングするために、ここで説明した方法を使用していました現像及び成人の脳20のその空間分布20を明らかにすることにより、この技術は、生物学的影響および5MCのこれらの修飾された誘導体は、細胞の分化、開発および疾患を含む検出することができ、様々な生物学的状況における5MCの酸化誘導体の運命を理解するために重要であることができます。また、ここで説明する方法は、tyramiと異なるインキュベーション時間で（染色の強度に比例する）、ペルオキシダーゼ反応の動態を評価することにより、異なる組織における5MCの酸化誘導体の半定量的な評価を与えることができますデ26。

DNA中のシトシン塩基（5MC）のメチル化は、1サイレンシング転写に関連した脊椎動物のゲノム中に見出される主要なエピジェネティックなマークを表します。 5MC導入及び維持DNAメチルトランスフェラーゼ2-5によって、ゲノムインプリンティング、X染色体不活性化、細胞の分化および発達3,6を含む生物学的プロセスの数に重要な役割を果たすことが示されているされている。従って、の破壊を5MCゲノムパターンは疾病7、8月11日の番号に関連付けられています。開発と疾患における5MCの役割を理解する上での進歩にもかかわらず、まだ、このマークを開発し、成体組織では削除されているかほとんどわかっていないされています。 15、14、13、DNA脱メチル化の潜在的なメカニズムは、最近12アクティブおよびパッシブ脱メチル化機構を含む提案されているいくつかの。十から十一の転座enzymによって媒介5MC逐次酸化の製品の発見5- hydroxylmethylcytosineとしてエス（tet1 / 2/3）（5hmC）、真核生物のDNA 16、17、18、19、5-formylcytosine（5FC）、および5-carboxylcytosine（5caC）は、それらの中間体として機能することができるかどうかを推測を促し自分の権利13におけるDNA脱メチル化のプロセスや行為などの安定したエピジェネティックなマーク。塩基除去修復の成分は、チミンDNAグリコシラーゼ（TDG）は、DNA 19から5FCと5caCの両方に結合し、除去することができることを実証しながら、20はアクティブなDNA脱メチル化における修飾5MC誘導体の役割を示唆しています。 5FC / 5caCが転写調節29におけるこれらのマークの潜在的な関与にRNA IIの処理能力の点の速度を調節することができることを示す最近の証拠。 5MCの酸化形態のこの潜在的な生物学的な重要性を、生化学的及び抗体ベースの技術の範囲は、それらのゲノム分布とグローバルコンテンツ16、19-24を研究するために使用されてきました。 トンのほとんどを考えます彼脊椎動物の器官は、異なる細胞型で構成され、修正されたシトシン塩基の分布は、組織及び細胞型特異16-18、20、23、25〜27であることが、種々の組織における酸化5MC誘導体の空間分布を決定することは重要な実験になりますそれらの生物学的機能を発表するために必要なタスク。生化学的および抗体ベースのアプローチのほとんどは、異なる組織および細胞型における5MCの修飾された形態の分布に関するあらゆる空間情報を提供していません。これとは対照的に、免疫化学ベースの技術は5MC 28とその酸化誘導体20の空間分布および核局在化を評価するための迅速なツールを提供することができます。それを言われて、マウスゲノム18における5FC（各10 6シトシン20）と5caC（各10 6シトシン3）の報告が非常に低い存在量は、標準的な免疫化学のための重要な課題です。 ここに、我々は、堅牢提供し、高感度免疫化学的方法および哺乳動物の脳組織中のシトシンの酸化型の迅速な検出を説明します。信号増幅ステップと結合されたペルオキシダーゼ結合二次抗体を組み込むことによって、この方法は、5FCと5caCの非常に少量を検出する課題を回避します。また、この技術は、効果的にこれらのエピジェネティックマークの生物学的機能を解明する他のアプローチを補完し、系統特異的マーカーとシトシンの修飾形態を同時検出するために用いることができます。

<table border="0" cellpadding="0" cellspacing="0" width="1281">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:388px;">Coplin jar</td>
			<td style="width:180px;">Cole-Parmer</td>
			<td style="width:119px;">UY-48585-30</td>
			<td style="width:595px;">Glass made</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Xylene</td>
			<td></td>
			<td></td>
			<td>Use only in Class II safety cabinet</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphate buffer saline (PBS)</td>
			<td>Fisher Scientific</td>
			<td>12821680</td>
			<td>pPH 7.5, filtered before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4% paraformaldehyde (PFA)</td>
			<td>Sigma Aldrich</td>
			<td>P6148</td>
			<td>Once made can be stored at -20&#176;C in aliquotes for several months</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4% formaldehyde&#160; (FA)</td>
			<td>Sigma Aldrich</td>
			<td>F8775</td>
			<td>Once made can be stored at -20&#176;C in aliquotes for several months</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethanol, anhydrous denatured, histological grade</td>
			<td>Sigma Aldrich</td>
			<td>&#160;64-17-5&#160;&#160;</td>
			<td>95, 75, 50 % in PBS</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.01 % Tween 20 in PBS</td>
			<td>Sigma Aldrich</td>
			<td>P9416</td>
			<td>PBT</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.5% Triton X-100 in PBS</td>
			<td>Sigma Aldrich</td>
			<td>X100</td>
			<td>PBX</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2 N HCl</td>
			<td>Sigma Aldrich</td>
			<td>71826</td>
			<td>caution extremely toxic</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">100 mM Tris-HCl&#160;</td>
			<td>Promega</td>
			<td>H5121</td>
			<td>PH adjusted to 8.5</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">hydrophobic barrier pen</td>
			<td>Abcam</td>
			<td>ab2601</td>
			<td>for immunohistochemistry</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Slide moisture chamber</td>
			<td>Scientific Device Laboratory</td>
			<td>197-BL</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-5mC mouse antibody</td>
			<td>Diagenode</td>
			<td>C15200081</td>
			<td>monoclonal primary antibody</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-5hmC antibody</td>
			<td>Active Motif</td>
			<td>39791</td>
			<td>rabbit polyclonal primary antibody</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-5caC antibody</td>
			<td>Active Motif</td>
			<td>61225</td>
			<td>rabbit polyclonal primary antibody</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Peroxidase-conjugated anti-rabbit seconday antibody</td>
			<td>Dako</td>
			<td>K1497</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">555-congjuated goat anti-mouse antibody</td>
			<td>Molecular probes</td>
			<td>A-11005</td>
			<td>&#160;secondary antibody</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10% BSA</td>
			<td>Sigma Aldrich</td>
			<td>A9418</td>
			<td>Blocking solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">22x32mm Glass cover slips</td>
			<td>BDH</td>
			<td>406/0188/24</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tyramide Signal Amplification System</td>
			<td>Perkin Elmer</td>
			<td>NEL741001KT</td>
			<td>Other fluorochome conjugated seconday antibodies can be used for co-detection of oxi-5mC&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#160;Mounting Medium with DAPI</td>
			<td>Vector Labs</td>
			<td>H-1200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Colourless nail polish</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">chicken polyclonal anti-GFAP polyclonal antibody</td>
			<td>Thermo Scientific</td>
			<td style="width:119px;">PA5-18598</td>
			<td>1:400 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-NeuN mouse monoclonal antibody</td>
			<td>Merck milipore</td>
			<td style="width:119px;">MAB377B</td>
			<td>1:400 dilution</td>
		</tr>
	</tbody>
</table>

detection of modified forms of cytosine using sensitive immunohistochemistry

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. 5-hydroxylmethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) are the recently discovered modified cytosine bases produced by enzymatic oxidation of 5mC, whose biological functions remain relatively obscure. A number of approaches ranging from biochemical to antibody based techniques have been employed to study the genomic distribution and global content of these modifications in various biological systems. Although some of these approaches can be useful for quantitative assessment of these modified forms of 5mC, most of these methods do not provide any spatial information regarding the distribution of these DNA modifications in different cell types, required for correct understanding of their functional roles. Here we present a highly sensitive method for immunochemical detection of the modified forms of cytosine. This method permits co-detection of these epigenetic marks with protein lineage markers and can be employed to study their nuclear localization, thus, contributing to deciphering their potential biological roles in different experimental contexts.

脳組織切片における5hmCの分布を決定するために、我々は、具体的には、このマークが相互作用するが市販の抗5hmC抗体を用いて、有糸分裂後ニューロン、NeuNのためのマーカーで、このエピジェネティック修飾の同時検出を行っていない変更された他の形態でシトシン20、25。成人の脳における5hmCと5caC分布の免疫組織化学的分析は、著名な5hmC染色がのNeuN陽性細胞との共局在のに対し、NeuNの陰性のグリア細胞は、ゲノム5hmC（ 図1）20のより低いレベルを有することが明らかになりました。 我々は最近のTet-依存5MC酸化が神経幹細胞（NSC）20の系統の仕様の間に動作可能であることを示しました。 NSCは、5FCと5caCに免疫化学的に検出不可能であるにもかかわらずニューロンのAに向けてのNSC分化の初期段階で顕著な免疫染色​​を示します<html> NDグリア系統。これらの両方のマークは、一過性の早期のニューロンとグリア分化20のマーカーの出現と同時に蓄積します。我々はグリア分化誘導の3日目にNSCの固定の文化にグリアマーカーGFAPと、このマークの共染色を行ったNSCを分化に5caCの分布を決定すること。 NSC又は成熟アストロサイトとは異なり（データは示さず）、我々は、これらの培養物においてGFAPを発現する細胞（ 図2）20の比較的大きな割合で強い5caCシグナルを観察しました。 <img alt="図1" src="/files/ftp_upload/54416/54416fig1.jpg" /> DAPI（青）で対比成人脳組織内のNeuN（赤）と5hmC（緑）の図1.共同免疫染色。個々のチャネルおよびマージされた図が示されています。スケールバーは25μmです。「ブランク&gt;この図の拡大版をご覧になるにはこちらをクリックしてください。 <img alt="図2" src="/files/ftp_upload/54416/54416fig2.jpg" /> グリア分化の3日目でのNSCの培養におけるGFAPと5caCの図2.共免疫染色。個々のチャネルおよびマージされた図が示されています。スケールバーは20μmである。 <a href="https://www.jove.com/files/ftp_upload/54416/54416fig2large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a>

Watch this Scientific Journal Video about Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry at JoVE.com

Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry

Herein we describe a sensitive immunochemical method for mapping the spatial distribution of 5mC oxidation derivatives based on the use of peroxidase-conjugated secondary antibodies and tyramide signal amplification.

高感度免疫組織化学を使用してシトシンの修飾された形態の検出

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. ...

detection-modified-forms-cytosine-using-sensitive

Research

JoVE Journal

Biology

7.2K ビュー.  Université Libre de Bruxelles. Herein we describe a sensitive immunochemical method for mapping the spatial distribution of 5mC oxidation derivatives based on the use of peroxidase-conjugated secondary antibodies and tyramide signal amplification.

ビデオ: Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry

Thin Sectioning of Slice Preparations for Immunohistochemistry

Many investigations in neuroscience, as well as other disciplines, involve studying small, yet macroscopic pieces or sections of tissue that have been preserved, freshly removed, or excised but kept viable, as in slice preparations of brain tissue.  Subsequent microscopic studies of this material can be challenging, as the tissue samples may be difficult to handle.  Demonstrated here is a method for obtaining thin cryostat sections of tissue with a thickness that may range from 0.2-5.0 mm.  We routinely cut 400 micron thick Vibratome brain slices serially into 5-10 micron coronal cryostat sections.  The slices are typically first used for electrophysiology experiments and then require microscopic analysis of the cytoarchitecture of the region from which the recordings were observed.  We have constructed a simple device that allows controlled and reproducible preparation and positioning of the tissue slice.  This device consists of a cylinder 5 cm in length with a diameter of 1.2 cm, which serves as a freezing stage for the slice.  A ring snugly slides over the cylinder providing walls around the slice allowing the tissue to be immersed in freezing compound (e.g., OCT).  This is then quickly frozen with crushed dry ice and the resulting  wafer  can be position easily for cryostat sectioning.  Thin sections can be thaw-mounted onto coated slides to allow further studies to be performed, such as various staining methods, in situ hybridization, or immunohistochemistry, as demonstrated here.

The present method allows reproducible cryostat sectioning of small, difficult-to-manage, tissue pieces, such as biopsies and brain slices. We utilize a simple aluminum freezing stage to facilitate handling of tissue and a standard cryostat to routinely produce 5-10 micron serial sections from 400 micron thick brain slices.

免疫組織化学のためのスライス標本の薄いセクショニング

Many investigations in neuroscience, as well as other disciplines, involve studying small, yet macroscopic pieces or sections of tissue that have been ...

生物学

Preventing the Spread of Malaria and Dengue Fever Using Genetically Modified Mosquitoes

In this candid interview, Anthony A. James explains how mosquito genetics can be exploited to control malaria and dengue transmission.   Population replacement strategy, the idea that transgenic mosquitoes can be released into the wild to control disease transmission, is introduced, as well as the concept of genetic drive and the design criterion for an effective genetic drive system. The ethical considerations of releasing genetically-modified organisms into the wild are also discussed.

In this candid interview, Anthony A. James explains how mosquito genetics can be exploited to control malaria and dengue transmission. Population replacement strategy, the idea that transgenic mosquitoes can be released into the wild to control disease transmission, is introduced as well as the concept of genetic drive and the design criterion for an effective genetic drive system. The ethical considerations of releasing genetically-modified organisms into the wild are also discussed.

遺伝子組換え蚊を使用してマラリアやデング熱の拡大防止

In this candid interview, Anthony A. James explains how mosquito genetics can be exploited to control malaria and dengue transmission.   Population ...

Immunohistochemistry: Paraffin Sections Using the Vectastain ABC Kit from Vector Labs

Immunohistochemistry (IHC) is a valuable technique utilized to localize/visualize protein expression in a mounted tissue section using specific antibodies. There are two methods: the direct and indirect method. In this experiment, we will only describe the use of indirect IHC staining. Indirect IHC staining utilizes highly specific primary and biotin-conjugated secondary antibodies. Primary antibodies are utilized to discretely identify proteins of interest by binding to a specific epitope, while secondary antibodies subtract for non-specific background staining and amplify signal by forming complexes to the primary antibody. Slides can either be generated from frozen sections, or paraffin embedded sections mounted on glass slides. In this protocol, we discuss the preparation of paraffin-embedded sections by dewaxing, hydration using an alcohol gradient, heat induced antigen retrieval, and blocking of endogenous peroxidase activity and non-specific binding sites. Some sections are then stained with antibodies specific for T cell marker CD8 and while others are stained for tyrosine hydroxylase. The slides are subsequently treated with appropriate secondary antibodies conjugated to biotin, then developed utilizing avidin-conjugated horseradish peroxidase (HRP) with Diaminiobenzidine (DAB) as substrate. Following development, the slides are counterstained for contrast, and mounted under coverslips with permount. After adequate drying, these slides are then ready for imaging.

免疫組織化学：ベクターLabsからベクタステインABCキットを用いたパラフィン切片

Immunohistochemistry (IHC) is a valuable technique utilized to localize/visualize protein expression in a mounted tissue section using specific ...

Immunohistochemistry on Paraffin Sections of Mouse Epidermis Using Fluorescent Antibodies

In the epidermis, immunohistochemistry is an efficient means of localizing specific proteins to their relative expression compartment; namely the basal, suprabasal, and stratum corneum layers. The precise localization within the epidermis of a particular protein lends clues toward its functional role within the epidermis. In this chapter, we describe a reliable method for immunolocalization within the epidermis modified for both frozen and paraffin sections that we use very routinely in our laboratory. Paraffin sections generally provide much better morphology, hence, superior results and photographs; however, not all antibodies will work with the harsh fixation and treatment involved in their processing. Therefore, the protocol for frozen sectioning is also included. Within paraffin sectioning, two fixation protocols are described (Bouin's and paraformaldehyde); the choice of fixative will be directly related to the antibody specifications and may require another fixing method.

We describe a reliable method for immunolocalization within the epidermis modified for both frozen and paraffin sections that we use very routinely in our laboratory.

蛍光抗体を用いたマウス表皮のパラフィン切片で免疫組織化学

In the epidermis, immunohistochemistry is an efficient means of localizing specific proteins to their relative expression compartment; namely the basal, ...

Drosophila Larval NMJ Immunohistochemistry

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 31 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. Immunohistochemistry can be used to clearly image the components of the NMJ. In this article, we demonstrate how to use antibody staining to visualize the Drosophila larval NMJ.

Drosophila Larval NMJ Immunohistochemistry

This protocol demonstrates how to perform immunohistochemistry on dissected Drosophila larva.

ショウジョウバエ幼虫NMJ免疫組織化学

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use ...

Optical Recording of Electrical Activity in Guinea-pig Enteric Networks using Voltage-sensitive Dyes

The enteric nervous system (ENS) is a self-contained network with identified functions, capable of performing complex behaviors in isolation. Its neurons (10 to 25 &mu;m in diameter) are arranged in plexuses that are confined to distinct planes of the gut wall 1; the myenteric plexus can be found between the longitudinal and circular muscle layers, and the submucous plexus between the circular muscle layer and the mucosa. Since the effector systems for these plexuses (transporting epithelium, endocrine cells, immune elements, blood vessels and smooth muscle) are also contained within the gut wall, semi-intact preparations can be dissected that preserve individual components of different reflex pathways. The behavior of the effector systems is controlled by the submucous and myenteric plexuses acting in concert. Therefore, detailed knowledge of synaptic interactions within and between ganglia, and of communication between the plexuses, is essential for understanding normal gastrointestinal function. The ENS, as an intact nervous system, is a unique experimental model in which one can correlate molecular and cellular events with the electrical behavior of the neuronal network and its physiological outputs. Because of the quasi-two-dimensional organization of its plexuses, the ENS is particularly well suited for the study of neural networks using multiple site optical recording techniques that employ voltage-sensitive dyes 2,7,8,9. We will illustrate here the use of a relatively new naphthylstyryl-pyridinium dye (di-4-ANEPPDHQ) 3 that offers multiple advantages over its predecessors, including very low phototoxicity, slow rate of internalization, and remarkable chemical stability. When used in conjunction with a camera that permits sub-millisecond time resolution, this dye allows us to monitor the electrical activity of all the neurons in the field of view with a maximal spatial resolution of ~ 2.5 &mu;m at 100X magnification. At lower magnification (10X or 20X), the sacrifice of single-cell resolution is compensated by a gain in perspective, revealing the intricacies of the inter-ganglionic circuitry.

This protocol illustrates how voltage-sensitive dyes enable optical recording of electrical activity from intact neural networks such as the plexuses of the guinea-pig enteric nervous system, with an adjustable resolution that ranges from single-cells to multi-ganglionic circuitry.

電位感受性色素を用いてモルモット腸ネットワークにおける電気活動の光学的記録

The enteric nervous system (ENS) is a self-contained network with identified functions, capable of performing complex behaviors in isolation. Its neurons ...

Drosophila Pupal Abdomen Immunohistochemistry

The Drosophila pupal abdomen is an established model system for the study of epithelial morphogenesis and the development of sexually dimorphic morphologies 1-3. During pupation, which spans approximately 96 hours (at 25 &deg;C), proliferating populations of imaginal cells replace the larval epidermis to generate the adult abdominal segments. These imaginal cells, born during embryogenesis, exist as lateral pairs of histoblast nests in each abdominal segment of the larvae. Four pairs of histoblast nests give rise to the adult dorsal cuticle (anterior and posterior dorsal nests), the ventral cuticle (ventral nests) and the spiracles associated with each segment (spiracle nests) 4. Upon puparation, these diploid cells (distinguishable by size from the larger polyploid larval epidermal cells- LECs) begin a stereotypical process of proliferation, migration and replacement of the LECs. Various molecular and genetic tools can be employed to investigate the contributions of genetic pathways involved in morphogenesis of the adult abdomen. Ultimate adult phenotypes are typically analyzed following dissection of adult abdominal cuticles. However, investigation of the underlying molecular processes requires immunohistochemical analyses of the pupal epithelium, which present unique challenges. Temporally dynamic morphogenesis and the interactions of two distinct epithelial populations (larval and imaginal) generate a fragile tissue prone to excessive cell loss during dissection and subsequent processing. We have developed methods of dissection, fixation, mounting and imaging of the Drosophila pupal abdominem epithelium for immunohistochemical studies that generate consistent high quality samples suitable for confocal or standard fluorescent microscopy.

Drosophila Pupal Abdomen Immunohistochemistry

Antibody staining of the Drosophila pupae can enhance genetic analyses of adult abdominal developmental genetics. We present our protocol for dissection, fixation and antibody staining of staged Drosophila pupal abdomen.

ショウジョウバエの蛹の腹部の免疫組織化学

The Drosophila pupal abdomen is an established model system for the study of epithelial morphogenesis and the development of sexually dimorphic ...

qPCR Is a Sensitive and Rapid Method for Detection of Cytomegaloviral DNA in Formalin-fixed, Paraffin-embedded Biopsy Tissue

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for developing severe infection. Many laboratory methods for the detection of CMV infection have been developed, including serology, viral culture, and molecular methods. Often, these methods reflect systemic involvement with CMV and do not specifically identify local tissue involvement. Therefore, detection of CMV infection in the GI tract is frequently done by traditional histology of biopsy tissue. Hematoxylin and eosin (H&#38;E) staining in conjunction with immunohistochemistry (IHC) have remained the mainstays of examining these biopsies. H&#38;E and IHC sometimes result in atypical (equivocal) staining patterns, making interpretation difficult. It was shown that quantitative polymerase chain reaction (qPCR) for CMV can successfully be performed on formalin-fixed, paraffin-embedded (FFPE) biopsy tissue for very high sensitivity and specificity. The goal of this protocol is to demonstrate how to perform qPCR testing for the detection of CMV in FFPE biopsy tissue in a clinical laboratory setting. This method is likely to be of great benefit for patients in cases of equivocal staining for CMV in GI biopsies.

This protocol describes qPCR detection of cytomegalovirus in formalin-fixed, paraffin-embedded biopsy tissue, which is rapid, sensitive, specific, and useful for interpreting equivocal hematoxylin and eosin or immunohistochemical staining patterns.

定量PCRは、ホルマリン固定、パラフィン包埋された生検組織中のサイトメガロウイルスDNAの検出のための高感度かつ迅速な方法であり、

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for ...

Colorimetric Detection of Bacteria Using Litmus Test

There are increasing demands for simple but still effective methods that can be used to detect specific pathogens for point-of-care or field applications. Such methods need to be user-friendly and produce reliable results that can be easily interpreted by both specialists and non-professionals. The litmus test for pH is simple, quick, and effective as it reports the pH of a test sample via a simple color change. We have developed an approach to take advantage of the litmus test for bacterial detection. The method exploits a bacterium-specific RNA-cleaving DNAzyme to achieve two functions: recognizing a bacterium of interest and providing a mechanism to control the activity of urease. Through the use of magnetic beads immobilized with a DNAzyme-urease conjugate, the presence of bacteria in a test sample is relayed to the release of urease from beads to solution. The released urease is transferred to a test solution to hydrolyze urea into ammonia, resulting in an increase of pH that can be visualized using the classic litmus test.

We describe a protocol for colorimetric detection of E. coli using a modified litmus test that takes advantage of an RNA-cleaving DNAzyme, urease, and magnetic beads.

リトマス試験を用いて細菌の比色検出

There are increasing demands for simple but still effective methods that can be used to detect specific pathogens for point-of-care or field applications. ...

Sensitive Measurement of Mitophagy by Flow Cytometry Using the pH-dependent Fluorescent Reporter mt-Keima

Mitophagy is a process of selective removal of damaged or unnecessary mitochondria using autophagy machinery. Close links have been found between defective mitophagy and various human diseases, including neurodegenerative diseases, cancer, and metabolic diseases. In addition, recent studies have shown that mitophagy is involved in normal cellular processes, such as differentiation and development. However, the precise role of and molecular mechanisms underlying mitophagy require further study. Therefore, it is critical to develop a robust and convenient method for measuring changes in mitophagy activity. Here, we describe a detailed protocol for quantitatively assessing mitophagy activity through flow cytometry using the mitochondria-targeted fluorescent protein Keima (mt-Keima). This flow cytometry assay can analyze mitophagy activity more rapidly and sensitively than conventional microscopy- or immunoblotting-based methods. This protocol can be applied to analyze mitophagy activity in various cell types.

Mitophagy, the selective degradation of mitochondria, has been implicated in mitochondrial homeostasis and is deregulated in various human diseases. However, convenient experimental methods for measuring mitophagy activity are very limited. Here, we provide a sensitive assay for measuring mitophagy activity using flow cytometry.