We thank Ueli Grossniklaus (University of Z&#252;rich) for technical and financial support. We are thankful to Valeria Gagliardini, Christof Eichenberger, Arturo Bolanos and Peter Kopf for general lab support. This research was funded by the University of Z&#252;rich, grants from the Swiss National Foundation to CB (31003A_130722) and Ueli Grossniklaus (31003A_141245 and 31003AB-126006), and the Agence Nationale de la Recherche to DG (Programme ANR-BLANC-2012).

手順は、 図1のワークフローに記載されており、組織の切開および埋め込 ​​みのためのセットアップを図2に示す。 1。組織固定<ol><li>氷の上で焼きたてのBVO固定液緩衝液を含むマイクロチューブに、20〜30心皮を集める。 </li><li>穏やかに室温で振とうしながら組織30分を修正します。 </li><li> 400×gで卓上マイクロ遠心1分で固定液に心皮を含むチューブを回転。 </li><li>慎重に固定液緩衝液を除去し、PBTの1ミリリットルを加え、氷上にチューブを置きます。 </li></ol> 2。解剖と埋め込み<ol><li>新たに作製した、5％アクリルアミドミックスのそれぞれ200μlで5エッペンドルフチュー​​ブを準備します。 </li><li> 5スーパーフロストを70％エタノールで前洗浄し、鉛筆で標識摺動調製する。 </li><li> 20％APSと氷上で20％のNaPSをそれぞれの1アリコートを解凍。 </li><li>カットエンドチップで行われ4-5の心皮を取るきれいなスライド上に、液体の過剰を取り除く。 </li><li>細い針で縦カットを行い、 図2に示すように胚珠の行を解放する心皮の壁を切り離し、PBS（10μL以下で）で覆うことにより、乾燥を避ける。 </li><li>すぐに、200μlのアクリルアミドミックスのアリコートと12μLのAPSを12μlのNAPを追加し、混合する。 </li><li>解剖した胚珠に活性化したアクリルアミドの30を添加する。 </li><li> 20×20ミリメートルのカバーガラスで覆い、室温、45〜60分で重合してみましょう。 </li><li>カミソリの刃を使用してカバーガラスを取り外します。この段階で、サンプルはPBSを含むコプリンジャー中で4℃で一晩維持することができる。 </li></ol> 3。組織処理注：3.2.1、3.2.3、3.3.2および3.4​​.3を除くすべてのステップは室温での化学ボンネットの下に80ミリリットル溶液をコプリンジャーに行われている。スライドはフラットチップピンセットで転送されます。 <ol><li>組織の明確化と固定： <ol><li>メタノール中で5分間インキュベートする。 </li><li>エタノールに5分間インキュベート。 </li><li>エタノール中で30分間インキュベートする：キシレン（1:1）。 </li><li>エタノールに5分間インキュベート。 </li><li>メタノール中で5分間インキュベートする。 </li><li> 2.5％ホルムアルデヒドで補完、メタノールおよびPBT（1:1）中で15分間インキュベートする。 </li><li> PBTで2×10分を洗浄します。この段階で、スライドを4℃で一晩保持することができる</li></ol></li><li>細胞壁消化： <ol><li>氷上で細胞壁消化混合物のアリコートを解凍する。 </li><li>コプリンジャーからスライドを取り、ペーパータオルの上に垂直に配置することによって、液体の過剰を排出。 </li><li>アクリルアミドパッド上で細胞壁の消化ミックスを100μlを加え、23 X 46ミリメートルのカバースリップでカバーしています。他のスライドについて、この手順を繰り返します。 （材料に記載されている）湿ったチャンバー内で37℃で2時間インキュベートする。 </li><li> PBTのスライド2×5分を洗ってください。 </li></ol></li><li>治療をRNアーゼ： <ol><li>コプリンジャーからスライドを取り、目のドレイン前のような液体のE過剰。 </li><li>湿ったチャンバー内で37℃で1時間、1％のTween-20を含むPBS中の100μg/ mlでのRNaseA100μlで各スライドをインキュベートします。 </li><li> PBTで2×5分間スライドを洗浄します。 </li></ol></li><li>ポスト固定および透過： <ol><li>新たに作成したPBT-Fでの20分間のポスト修正。 </li><li> PBTで10分間スライドを洗浄します。 </li><li> 4℃で2％のTween-20を含むPBS中で2時間透過化</li><li> PBTで2×5分間スライドを洗浄します。 </li></ol></li></ol> 4。免疫染色注：このステップでは、一次抗体の最適濃度は、抗体の異なる希釈（1:200、1:500、1:1000）を用いて試験されなければならない。 <ol><li> 4℃で12〜24時間、0.2％のTween-20を含むPBSで希釈した一次抗体100μlで各スライドをインキュベート</li><li>穏やかに振とう下、室温で2〜4時間、PBTでスライドを洗浄します。 </li><li>適用するPBS +0.2％のTween-20で24時間、4℃で、二次抗体1:200 </li><li>穏やかに振とう下、室温で1時間、PBTでスライドを洗浄します。 </li><li> 15分間、PBS中10μg/ mlのヨウ化と対比染色後、室温で、穏やかに振盪下にPBS中で15分をすすぐ。 </li><li>退色防止液体封入内のマウントは、10μg/ mlのヨウ化を補った。封入剤は、CLSMで画像を取得する前に、1時間硬化させてみましょう。 </li></ol> 5。定量的イメージング<ol><li>画像収集： <ol><li>理想的には、より良好な長期のイメージング23を超える蛍光シグナルの保存、および63Xグリセロール浸レンズを可能にする共振型走査モードを使用して、CLSMを用いた高解像度の画像を取得する。 </li><li>厳密に従うことを通して、標準的な取得手順を定義するためにこのような実験の開始時レーザー強度、利得、ピンホール、ボクセルサイズとズーム倍率として取得パラメータをテスト一貫性の定量的測定のためのすべてのスライド。 </li><li>蛍光色素間のクロストークが存在しないことを確認してください。存在する場合、シーケンシャルスキャンを設定します。同時に、別々に透過画像を取得していない。 </li><li> xとyの次元で可能な限り高い分解能でかつZ次元（ナイキストの法則）での2倍のオーバーサンプリングをシリアル、3次元画像取得を実行します。 </li></ol></li><li>画像処理： <ol><li>商用またはオープンソースのソフトウェアを使用して3次元的に連続画像を再構成する。 </li><li> 3Dへの関心のそれぞれの核（またはセル）の周囲に輪郭面を定義します。 </li><li>各オブジェクト内の画素強度の和として、各チャンネルで蛍光を定量する。 </li><li>統計分析のためにデータをExcelにエクスポートします。 例えば、DNA染色信号に対する抗体シグナルを正規化する。 </li></ol></li></ol>

<ol>
	<li>Maheshwari, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> An introduction to the embryology of angiosperms. , (1950).</li><li>Schmidt, 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=Transcriptome+analysis+of+the+Arabidopsis+megaspore+mother+cell+uncovers+the+importance+of+RNA+helicases+for+plant+germline+development.">Transcriptome analysis of the Arabidopsis megaspore mother cell uncovers the importance of RNA helicases for plant germline development.</a> PLoS Biol. 9 (9), (2011).</li><li>Schmidt, M. 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=A+powerful+method+for+transcriptional+profiling+of+specific+cell+types+in+eukaryotes:+laser-assisted+microdissection+and+RNA+Sequencing.">A powerful method for transcriptional profiling of specific cell types in eukaryotes: laser-assisted microdissection and RNA Sequencing.</a> PLoS One. 7 (1), (2012).</li><li>Wuest, S. 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=Arabidopsis+female+gametophyte+gene+expression+map+reveals+similarities+between+plant+and+animal+gametes.">Arabidopsis female gametophyte gene expression map reveals similarities between plant and animal gametes.</a> Curr Biol. 20 (6), 506-512 (2010).</li><li>Koornneef, 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=Cytogenetic+tools+for+Arabidopsis+thaliana.">Cytogenetic tools for Arabidopsis thaliana.</a> Chromosome Res. 11 (3), 183-194 (2003).</li><li>Oliver, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dynamics+of+histone+H3+modifications+is+species-specific+in+plant+meiosis.">The dynamics of histone H3 modifications is species-specific in plant meiosis.</a> Planta. 238 (1), 23-33 (2013).</li><li>Ravi, 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=Meiosis-specific+loading+of+the+centromere-specific+histone+CENH3+in+Arabidopsis+thaliana.">Meiosis-specific loading of the centromere-specific histone CENH3 in Arabidopsis thaliana.</a> PLoS Genet. 7 (6), (2011).</li><li>Borges, 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=Reprogramming+the+epigenome+in+Arabidopsis+pollen.">Reprogramming the epigenome in Arabidopsis pollen.</a> Cold Spring Harb Symp Quant Biol. 77, 1-5 (2012).</li><li>Pandey, P., 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=Chromatin+alterations+during+pollen+development+in+Hordeum+vulgare.">Chromatin alterations during pollen development in Hordeum vulgare.</a> Cytogenet Genome Res. 141 (1), 50-57 (2013).</li><li>Schoft, V. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+RNA-directed+DNA+methylation+upon+decondensation+of+constitutive+heterochromatin.">Induction of RNA-directed DNA methylation upon decondensation of constitutive heterochromatin.</a> EMBO Rep. 10 (9), 1015-1021 (2009).</li><li>Barrell, P. J., Grossniklaus, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confocal+microscopy+of+whole+ovules+for+analysis+of+reproductive+development:+the+elongate1+mutant+affects+meiosis+II.">Confocal microscopy of whole ovules for analysis of reproductive development: the elongate1 mutant affects meiosis II.</a> Plant J. 43 (2), 309-320 (2005).</li><li>Braselton, J. P., 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=Feulgen+staining+of+intact+plant+tissues+for+confocal+microscopy.">Feulgen staining of intact plant tissues for confocal microscopy.</a> Biotech. Histochem. 71, 84-87 (1996).</li><li>Scott, R. 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=Parent-of-origin+effects+on+seed+development+in+Arabidopsis+thaliana.">Parent-of-origin effects on seed development in Arabidopsis thaliana.</a> Development. 125, 3329-3341 (1998).</li><li>Armstrong, S. J., Jones, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Female+meiosis+in+wild-type+Arabidopsis+thaliana+and+in+two+meiotic+mutants.">Female meiosis in wild-type Arabidopsis thaliana and in two meiotic mutants.</a> Sex Plant Reprod. 13, 177-183 (2001).</li><li>Grimanelli, 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=Heterochronic+expression+of+sexual+reproductive+programs+during+apomictic+development+in+Tripsacum.">Heterochronic expression of sexual reproductive programs during apomictic development in Tripsacum.</a> Genetics. 165, 1521-1531 (2003).</li><li>Gutierrez-Marcos, J. 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=Maternal+gametophytic+baseless1+is+required+for+development+of+the+central+cell+and+early+endosperm+patterning+in+maize+(Zea+mays).">Maternal gametophytic baseless1 is required for development of the central cell and early endosperm patterning in maize (Zea mays).</a> Genetics. 174 (1), 317-329 (2006).</li><li>Niedojad&#322;o, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribosomal+RNA+of+Hyacinthus+orientalis+L.+female+gametophyte+cells+before+and+after+fertilization.">Ribosomal RNA of Hyacinthus orientalis L. female gametophyte cells before and after fertilization.</a> Planta. 236, 171-184 (2012).</li><li>Williams, J. H., Friedman, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+four-celled+female+gametophyte+of+Illicium+(Illiciaceae;+Austrobaileyales):+Implications+for+understanding+the+origin+and+early+evolution+of+monocots,+eumagnoliids,+and+eudicots.">The four-celled female gametophyte of Illicium (Illiciaceae; Austrobaileyales): Implications for understanding the origin and early evolution of monocots, eumagnoliids, and eudicots.</a> Am J Bot. 91 (3), 332-351 (2004).</li><li>Bass, H. 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=Telomeres+cluster+de+novo+before+the+initiation+of+synapsis:+a+three-dimensional+spatial+analysis+of+telomere+positions+before+and+during+meiotic+prophase.">Telomeres cluster de novo before the initiation of synapsis: a three-dimensional spatial analysis of telomere positions before and during meiotic prophase.</a> J Cell Biol. 137 (1), 5-18 (1997).</li><li>Howe, E. 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=Three-dimensional+acrylamide+fluorescence+in+situ+hybridization+for+plant+cells.">Three-dimensional acrylamide fluorescence in situ hybridization for plant cells.</a> Methods Mol Biol. 990, 53-66 (2013).</li><li>She, 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=Chromatin+reprogramming+during+the+somatic-to-reproductive+cell+fate+transition+in+plants.">Chromatin reprogramming during the somatic-to-reproductive cell fate transition in plants.</a> Development. 140 (19), 4008-4019 (2013).</li><li>Pillot, 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=Embryo+and+endosperm+inherit+distinct+chromatin+and+transcriptional+states+from+the+female+gametes+in+Arabidopsis.">Embryo and endosperm inherit distinct chromatin and transcriptional states from the female gametes in Arabidopsis.</a> Plant Cell. 22 (2), 307-320 (2010).</li><li>Borlinghaus, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MRT+letter:+high+speed+scanning+has+the+potential+to+increase+fluorescence+yield+and+to+reduce+photobleaching.">MRT letter: high speed scanning has the potential to increase fluorescence yield and to reduce photobleaching.</a> Microsc Res Tech. 69 (9), 689-692 (2006).</li><li>Autran, 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=Maternal+epigenetic+pathways+control+parental+contributions+to+Arabidopsis+early+embryogenesis.">Maternal epigenetic pathways control parental contributions to Arabidopsis early embryogenesis.</a> Cell. 145 (5), 707-719 (2011).</li><li>Fransz, P., 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=Interphase+chromosomes+in+Arabidopsis+are+organized+as+well+defined+chromocenters+from+which+euchromatin+loops+emanate.">Interphase chromosomes in Arabidopsis are organized as well defined chromocenters from which euchromatin loops emanate.</a> Proc Natl Acad Sci USA. 99 (22), 14584-14589 (2002).</li><li>Cox, W. G., Singer, V. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+DNA+hybridization+probe+preparation+using+amine+modification+and+reactive+dye+coupling.">Fluorescent DNA hybridization probe preparation using amine modification and reactive dye coupling.</a> BioTechniques. 36 (1), 114-122 (2004).</li><li>Lysak, M., Salinas, J., Sanchez-Serrano, J. 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=.">.</a> Cytogenetic analyses of Arabidopsis In Arabidopsis Protocols: Methods in Molecular Biology, 2nd ed. , 173-186 (2006).</li><li>Chieco, P., Derenzini, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Feulgen+reaction+75+years+on.">The Feulgen reaction 75 years on.</a> Histochem Cell Biol. 111 (5), 345-358 (1999).</li><li>Suzuki, T., 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+staining+for+fluorescence+and+laser+confocal+microscopy.">DNA staining for fluorescence and laser confocal microscopy.</a> J Histochem Cytochem. 45 (1), 49-53 (1997).</li><li>Egelhofer, T. 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=An+assessment+of+histone-modification+antibody+quality.">An assessment of histone-modification antibody quality.</a> Nat Struct Mol Biol. 18 (1), 91-93 (2011).</li><li>Bauwens, S., Oostveldt, P. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+mount+fluorescence+in+situ+hybridization+(FISH)+of+repetitive+DNA+sequences+on+interphase+nuclei+of+the+small+cruciferous+plant+Arabidopsis+thaliana.">Whole mount fluorescence in situ hybridization (FISH) of repetitive DNA sequences on interphase nuclei of the small cruciferous plant Arabidopsis thaliana.</a> Procedures for In Situ Hybridization to Chromosomes, Cells, and Tissue Sections. Nonradioactive in situ hybridization application manual. , 165-171 (1996).</li></ol>

The authors declare that they have no competing financial interests.

顕花植物では、女性の生殖細胞系列は、このように技術的に困難なホールマウントで細胞学的染色をレンダリング、珠心や胚珠外皮を含むいくつかの細胞層に囲まれています。ここでは、このような全体マウントでのin situハイブリダイゼーション 、免疫染色、DNA染色および蛍光として細胞学的染色に適し胚珠多数の製造および加工を可能に効率的なプロトコルを提示する。我?...

顕花植物では、生殖系統の確立は、SMCの分化、女性MMCおよび男性の胞子母細胞から始まります。 MMCは胚珠原基の遠位先端のサブ表皮珠心細胞から発達し、小胞子母細胞は、深い花器官1の内部に配置されている葯房に胞子形成組織から開発しています。平滑筋細胞は、その後、有糸分裂の際に配偶体を生じる一倍体胞子を生成する減数分裂を受ける。雌性配偶体、または胚嚢は、1卵細胞、1中央のセル、2助細胞と3 antipodalsで構成されています。雄性配偶体または花粉は一つの栄養細胞二つ精子細胞から構成されている。雄性配偶体は、比較的アクセス可能なオブジェクト·オブ·研究したまま雌性配偶体が胚珠内に埋め込まれ、それ自体が花の心皮で囲まれ​​、したがって、分子·細胞学的分析に固有の課題を提起する。しかし、近年、レーザ支援マイクロダイセクションは、トランスクリプトームは、MMCと女性の配偶体細胞2-4で分析することができ、エレガントなソリューションを提供しました。候補遺伝子の発現に加えてin situハイブリダイゼーションまたはレポーター遺伝子アッセイにおいて、例えば RNAを用いて、分析において、細胞学的分析は、特定の直接的な細胞染色または間接的な免疫染色を用いて内因性の細胞成分の動態を調査することができる。特に、一緒にクロマチンの修正やクロマチンの構成要素の免疫染色で、魚やDNAの染色を用いて細胞遺伝学的染色は、 シロイヌナズナ 5クロマチン動態と核組織を解明するための中心的なアプローチである。一般的に、減数分裂がよく、植物の雄減数母細胞6,7で検討されている特定の染色体動態を伴う。おそらく動的なエピジェネティックなリプログラミングを反映し、さらに大規模、細胞特異的なクロマチン再編成は、花粉の発達8月10日の間に記載されている。これとは対照的に、原因女性の減数分裂細胞と配偶体の相対的な到達不能に、これらの調査は、適用が技術的に困難で残っており、多くの場合（下記参照）切片または手動切開および酵素消化を必要とする。また、全体のマウントで光学的透明度の欠如は、優勢無傷胚珠の生殖細胞の高分解能撮像の障害となる。 ホールマウント胚珠における染色体組織の細胞学的分析のための古典的な方法は、フォイルゲン染色の11から13を使用しています。これは、タンパク質の変性をもたらすので、クロマチン構造の破壊を引き起こすDNAの（次亜塩素酸を用いて）酸加水分解を含む。代わりに、女性の減数母細胞と配偶体細胞における染色体組織が ​​（例えば14〜18を参照）は、半薄切片または解剖し、胚嚢とMMCにDAPI染色および免疫染色を用いて観察することができます。明らかに、しかし、手動の解剖と分割は、労働集約的であることができ、クロマチンエピトープの多数の定性的および定量的分析に妨げる。 ここでは、全体のマウントの下流細胞学的染色に適した種々のシロイヌナズナ胚珠を多数準備するために効率的なプロトコルを提供する。簡単に説明すると、花芽が固定液中でインキュベートされる、胚珠の行は心皮から解剖され、花粉減数母細胞19,20のために行ったように、スライドにアクリルアミドに包埋した。埋め込まれた胚珠は、さらに細胞壁消化および透過化の前に、メタノール、エタノール、キシレン中でクリアされ、固定されている。これらのステップの可能なバリエーションが議論されている。次いで、試料をDNA染色、免疫染色およびFISHのために使用することができる。準備モードは、効率的で、並列実験設備（最大16のスライドが異なる下流の解析のために一日に製造することができる）が可能になります。記載され治療は、ホールマウントで均質なイネーブル信号を、よく組織学的、細胞の保存生殖細胞および細胞型の間の定性的および定量的な比較に利益をもたらす、周囲の珠心細胞および核の組織。校正され、3次元再構成に続いて、CLSMベースの高分解能イメージング、蛍光シグナルの意味のある定量的な測定を可能にします。我々が正常に差別MMC 21にクロマチン動態を解析するために、この手順を使用し、雌性配偶体22を開発。ここではホールマウント胚珠内のヘテロクロマチン解析、クロマチン免疫染色、GFP免疫染色およびFISHの代表的な結果を提示する。今後も我々のプロトコルは、他の植物組織および種に適していると考えています。

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an efficient method for quantitative, single-cell analysis of chromatin modification and nuclear architecture in whole-mount ovules in arabidopsis

植物の開花では、体への生殖細胞の運命遷移は大人の植物の花器官における胞子母細胞（SMC）の仕様でマークされています。女性のSMC（大胞子母細胞、MMC）は胚珠原基に区別し、減数分裂を受ける。選択された一倍体大胞子は、その後一緒にアクセサリー細胞と、配偶子を生じるであろう多雌性配偶体、卵細胞と中央のセルを形成するために、有糸分裂を受ける。胚珠内部MMC、減数分裂細胞と雌性配偶体の限られたアクセス性細胞学的および細胞遺伝学的ための技術的に困難である単一細胞レベルで分析する。特に、携帯電話または核エピトープの直接的または間接的な免疫が植物細胞および単一細胞イメージング内部の試薬の貧弱な浸透によって損なわれては、ホールマウント組織における光学的透明性の欠如によってdemisedされる。 そこで、NUCLを分析する効率的な方法を開発したホールマウント組み込みシロイヌナズナ胚珠内の単一のセルの高解像度で耳の組織とクロマチン修飾。これは、顕微鏡スライド上のアクリルアミドゲルの薄層内の固定胚珠の解剖と埋め込みに基づいています。埋め込まれた胚珠は、免疫染色試薬、組織の透明度および透過性を向上させることを目指した化学的および酵素的処理に供される。これらの治療は、細胞のクロマチン組織、DNAおよびタンパク質のエピトープを保持します。サンプルは、クロマチン免疫染色、in situハイブリダイゼーション （FISH）、 蛍光 、およびヘテロクロマチンの解析のためのDNA染色を含む種々の下流の細胞学的分析のために使用することができる。 3D再構成に続いて、高解像度の共焦点レーザー走査顕微鏡（CLSM）イメージングは​​、単細胞の解像度で定量的な測定を可能にする。

我々は全体のマウント中の細胞学的染色に適しシロイヌナズナ胚珠の大規模調製および処理のための堅牢なプロトコルを提供する。埋め込 ​​みのおかげで、胚珠が3次元構造（ 図3）を保持する。さらに、光の明確化など、組織処理は、高解像度で細胞内構造を画像化することができます。 図4は、ヘテロクロマチンが明るく表示され、明確に定義された目立...

Watch this Scientific Journal Video about An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis at JoVE.com

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

私たちは、免疫染色、蛍光in situハイブリダイゼーション 、ホールマウントシロイヌナズナ胚珠の量的、高分解能イメージングに続いてDNA染色のためにここに効率的で信頼性の高いプロトコルを提供する。この方法は、正常なクロマチン修飾および核構造を分析するために使用した。

ホールマウント胚珠内でのクロマチン修飾や核構造の定量的、単一細胞分析のための効率的な方法<em&gt;シロイヌナズナ</em

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the ...

an-efficient-method-for-quantitative-single-cell-analysis-chromatin

Research

JoVE Journal

Biology

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

13.1K ビュー.  University of Zürich. 私たちは、免疫染色、蛍光in situハイブリダイゼーション 、ホールマウントシロイヌナズナ胚珠の量的、高分解能イメージングに続いてDNA染色のためにここに効率的で信頼性の高いプロトコルを提供する。この方法は、正常なクロマチン修飾および核構造を分析するために使用した。 

ビデオ: An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

初期のニワトリ胚のin situハイブリダイゼーションダブルホールマウント

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

生物学

Method for Whole Mount Antibody Staining in Chick

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

チックの全マウントの抗体染色のための方法

Single-cell Suction Recordings from Mouse Cone Photoreceptors

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment are open and allow cations to flow steadily inwards across the membrane, depolarizing the cell. Light exposure triggers the closure of the CNG channels, blocks the inward cation current flow, and thus results in cell hyperpolarization. Based on the polarity of photoreceptors, a suction recording method was developed in 1970s that, unlike the classic patch-clamp technique, does not require penetrating the plasma membrane 1. Drawing the outer segment into a tightly-fitting glass pipette filled with extracellular solution allows recording the current changes in individual cells upon test-flash exposure. However, this well-established "outer-segment-in (OS-in)" suction recording is not suitable for mouse cone recordings, because of the low percentage of cones in the mouse retina (3%) and the difficulties in identifying the cone outer segments. Recently, an inner-segment-in (IS-in) recording configuration was developed to draw the inner segment/nuclear region of the photoreceptor into the recording pipette 2,3. In this video, we will show how to record from individual mouse cone photoresponses using single-cell suction electrode.

We will show how to record flash responses from single mouse cones using a suction electrode.

マウスの錐体光受容体からの単一細胞サクション録音

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

蛍光を用いたmRNA分子の可視化と分析<em&gt;その場で</emで&gt;ハイブリダイゼーション<em&gt;サッカロミセス·セレビシエ（Saccharomyces cerevisiae）</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ binding proteins that initiate Ca2+ signaling cascades. However, we still know little about how stimulus specific Ca2+ signals are generated. The specificity of a Ca2+ signal may be attributed to the sophisticated regulation of the activities of Ca2+ channels and/or transporters in response to a given stimulus. To identify these cellular components and understand their functions, it is crucial to use systems that allow a sensitive and robust recording of Ca2+ signals at both the tissue and cellular levels. Genetically encoded Ca2+ indicators that are targeted to different cellular compartments have provided a platform for live cell confocal imaging of cellular Ca2+ signals. Here we describe instructions for the use of two Ca2+ detection systems: aequorin based FAS (film adhesive seedlings) luminescence Ca2+ imaging and case12 based live cell confocal fluorescence Ca2+ imaging. Luminescence imaging using the FAS system provides a simple, robust and sensitive detection of spatial and temporal Ca2+ signals at the tissue level, while live cell confocal imaging using Case12 provides simultaneous detection of cytosolic and nuclear Ca2+ signals at a high resolution.

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Ca2+ signaling regulates diverse biological processes in plants. Here we present approaches for monitoring abiotic stress induced spatial and temporal Ca2+ signals in Arabidopsis cells and tissues using the genetically encoded Ca2+ indicators Aequorin or Case12.

時空のCaを測定する<sup&gt; 2 +</supシロイヌナズナ植物における&gt;シグナル

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ ...

A Whole Mount In Situ Hybridization Method for the Gastropod Mollusc Lymnaea stagnalis

Whole mount in situ hybridization (WMISH) is a technique that allows for the spatial resolution of nucleic acid molecules (often mRNAs) within a &#39;whole mount&#39; tissue preparation, or developmental stage (such as an embryo or larva) of interest. WMISH is extremely powerful because it can significantly contribute to the functional characterization of complex metazoan genomes, a challenge that is becoming more of a bottleneck with the deluge of next generation sequence data. Despite the conceptual simplicity of the technique much time is often needed to optimize the various parameters inherent to WMISH experiments for novel model systems; subtle differences in the cellular and biochemical properties between tissue types and developmental stages mean that a single WMISH method may not be appropriate for all situations. We have developed a set of WMISH methods for the re-emerging gastropod model Lymnaea stagnalis that generate consistent and clear WMISH signals for a range of genes, and across all developmental stages. These methods include the assignment of larvae of unknown chronological age to an ontogenetic window, the efficient removal of embryos and larvae from their egg capsules, the application of an appropriate Proteinase-K treatment for each ontogenetic window, and hybridization, post-hybridization and immunodetection steps. These methods provide a foundation from which the resulting signal for a given RNA transcript can be further refined with probe specific adjustments (primarily probe concentration and hybridization temperature).

A Whole Mount In Situ Hybridization Method for the Gastropod Mollusc Lymnaea stagnalis

The goal of this protocol is to provide users with a set of methods for the high-throughput decapsulation of Lymnaea stagnalis embryos and larvae in preparation for whole mount in situ hybridization, and for subsequent pre- and post-hybridization treatments.

全体のマウント<em&gt;その場で</em腹足類軟体動物のための&gt;ハイブリダイゼーション法<em&gt;モノアラガイstagnalis</em

Whole mount in situ hybridization (WMISH) is a technique that allows for the spatial resolution of nucleic acid molecules (often mRNAs) within a ...

CUBIC Protocol Visualizes Protein Expression at Single Cell Resolution in Whole Mount Skin Preparations

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium covering most of our body, and epidermal appendages such as the hair follicles and sweat glands. The epidermis undergoes regeneration throughout life and in response to injury. This is enabled by K14-expressing basal epidermal stem/progenitor cell populations that are tightly regulated by multiple regulatory mechanisms active within the epidermis and between epidermis and dermis. This article describes a simple method to clarify full thickness mouse skin biopsies, and visualize K14 protein expression patterns, Ki67 labeled proliferating cells, Nile Red labeled sebocytes, and DAPI nuclear labeling at single cell resolution in 3D. This method enables accurate assessment and quantification of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines. The CUBIC protocol is the best method available to date to investigate molecular and cellular interactions in full thickness skin biopsies at single cell resolution.

This report describes a CUBIC protocol to clarify full thickness mouse skin biopsies, and visualize protein expression patterns, proliferating cells, and sebocytes at the single cell resolution in 3D. This method enables accurate assessment of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines.

CUBICプロトコルは、全マウントスキン準備中の単一細胞の解像度でのタンパク質発現を可視化

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium ...

Visualization of 3D White Adipose Tissue Structure Using Whole-mount Staining

Adipose tissue is an important metabolic organ with high plasticity and is responsive to environmental stimuli and nutrient status. As such, various techniques have been developed to study the morphology and biology of adipose tissue. However, conventional visualization methods are limited to studying the tissue in 2D sections, failing to capture the 3D architecture of the whole organ. Here we present whole-mount staining, an immunohistochemistry method that preserves intact adipose tissue morphology with minimal processing steps. Hence, the structures of adipocytes and other cellular components are maintained without distortion, achieving the most representative 3D visualization of the tissue. In addition, whole-mount staining can be combined with lineage tracing methods to determine cell fate decisions. However, this technique has some limitations to providing accurate information regarding deeper parts of adipose tissue. To overcome this limitation, whole-mount staining can be further combined with tissue clearing techniques to remove the opaqueness of tissue and allow for complete visualization of entire adipose tissue anatomy using light-sheet fluorescent microscopy. Therefore, a higher resolution and more accurate representation of adipose tissue structures can be captured with the combination of these techniques.

The focus of the present study is to demonstrate the whole-mount immunostaining and visualization technique as an ideal method for 3D imaging of adipose tissue architecture and cellular component.

全取り付ける染色を用いた 3 D 白色脂肪組織構造の可視化

Adipose tissue is an important metabolic organ with high plasticity and is responsive to environmental stimuli and nutrient status. As such, various ...

An Easy and Flexible Inoculation Method for Accurately Assessing Powdery Mildew-Infection Phenotypes of Arabidopsis and Other Plants

Reducing crop losses due to fungal diseases requires improved understanding of the mechanisms governing plant immunity and fungal pathogenesis, which in turn requires accurate determination of disease phenotypes of plants upon infection with a particular fungal pathogen. However, accurate disease phenotyping with unculturable biotrophic fungal pathogens such as powdery mildew is not easy to achieve and can be a rate-limiting step of a research project. Here, we have developed a safe, efficient, and easy-to-operate disease phenotyping system using the Arabidopsis-powdery mildew interaction as an example. This system mainly consists of three components: (i) a wooden inoculation box fitted with a removable lid mounted with a stainless steel or nylon mesh of ~50 &#181;m pores for inoculating a flat of plants with fungal spores, (ii) a transparent plastic chamber with a small front opening for minimizing spore escape while conducting inoculation inside, and (iii) a spore-dislodging and distribution method for even and effective inoculation. The protocols described here include the steps and parameters for making the inoculation box and the plastic chamber at a low cost, and a video demonstration of how to use the system to enable even inoculation with powdery mildew spores, thereby improving accuracy and reproducibility of disease phenotyping.

We present a protocol for constructing a simple spore-distribution system consisting of an inoculation box with a ~50 &#181;m mesh and a transparent plastic chamber. This can be used to evenly inoculate plants with powdery mildew spores, thereby enabling accurate and reproducible assessment of disease phenotypes of plants under study.

シロイヌナズナなどのうどんこ病感染表現型を正確に評価するための簡便かつ柔軟な接種法

Reducing crop losses due to fungal diseases requires improved understanding of the mechanisms governing plant immunity and fungal pathogenesis, which in ...

Double-Staining Method to Detect Pectin in Plant-Fungus Interaction

Plant cells use different structural mechanisms, either constitutive or inducible, to defend themselves from fungal infection. Encapsulation is an efficient inducible mechanism to isolate the fungal haustoria from the plant cell protoplast. Conversely, pectin, one of the polymeric components of the cell wall, is a target of several pectolytic enzymes in necrotrophic interactions. Here, a protocol to detect pectin and fungal hyphae through optical microscopy is presented. The pectin-rich encapsulation in the cells of coffee leaves infected by the rust fungus Hemileia vastatrix and the mesophyll cell wall modification induced by Cercospora coffeicola are investigated. Lesioned leaf samples were fixed with the Karnovsky solution, dehydrated, and embedded in glycol methacrylate for 2-4 days. All steps were followed by vacuum-pumping to remove air in the intercellular spaces and improve the embedding process. The embedded blocks were sectioned into 5-7 &#181;m thick sections, which were deposited on a glass slide covered with water and subsequently heated at 40 &#176;C for 30 min. Next, the slides were double-stained with 5% cotton blue in lactophenol to detect the fungus and 0.05% ruthenium red in water to detect pectin (acidic groups of polyuronic acids of pectin). Fungal haustoria of Hemileia vastatrix were found to be encapsulated by pectin. In coffee cercosporiosis, mesophyll cells exhibited dissolution of cell walls, and intercellular hyphae and conidiophores were observed. The method presented here is effective to detect a pectin-associated response in the plant-fungi interaction.

This protocol describes a microscopical method to detect pectin in coffee-fungus interaction.