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).

절차는 그림 1에서 워크 플로에 ​​설명하고, 해부 및 조직의 매립에 대한 설정은 그림 2에 나타내었다. 1. 조직 고정 <ol><li> 얼음에 갓 만든 BVO 정착액 버퍼를 포함하는 미세 원심 분리 튜브에 20 ~ 30 심피를 수집합니다. </li><li> 부드러운 실온에서 진탕 조직 30 분 수정. </li><li> 400 X g에서 벤치 탑 마이크로 원심 1 분에 정착의 심피가 들어있는 튜브를 스핀. </li><li> 주의 깊게 정착 버퍼를 제거하고 PBT 1 ㎖​​를 추가, 얼음에 튜브를 배치합니다. </li></ol> 2. 해부 및 포함 <ol><li> 갓 만든, 5 %의 아크릴 아미드의 혼합 각각 200 ㎕ 씩 다섯 에펜 도르프 튜브를 준비합니다. </li><li> 다섯 Superfrost 슬라이드를 70 % 에탄올로 미리 청소하고 연필로 표시를 준비합니다. </li><li> 한 20 % APS의 나누어지는 얼음에 20 % 낮잠 각을 해동. </li><li> 컷 엔드 팁, 장소 4-5 심피을깨끗한 슬라이드에 그들, 액체의 초과를 제거합니다. </li><li> 미세 바늘 길이 상처를 확인하고 그림과 그림 2와 난자의 행을 해제 할 심피 벽을 분리, PBS (이하 10 μL)로 덮어서 건조하지. </li><li> 빨리 12 ㎕의 낮잠, 200 ㎕의 아크릴 아미드 믹스의 나누어지는 12 μL APS를 추가하고 혼합한다. </li><li> 해부 난자에 활성화 된 아크릴 아미드의 30 μl를 추가합니다. </li><li> 20 X 20mm의 커버 슬립 커버, 실내 온도, 45 ~ 60 분에서 중합 할 수 있습니다. </li><li> 면도날을 사용하여 커버 슬립을 제거합니다. 이 단계에서, 샘플은 PBS를 포함하는 코 플린 항아리에서 4 ° C에서 하룻밤 동안 유지 될 수있다. </li></ol> 3. 조직 처리 참고 : 3.2.1, 3.2.3, 3.3.2과 3.4.3를 제외한 모든 단계는 상온에서 화학 후드 아래에 80 ㎖의 용액으로 코 플린 단지에서 실시된다. 슬라이드에는 평면 팁 집게로 전송됩니다. <ol><li> 조직의 설명과고정 : <ol><li> 메탄올에서 5 분 알을 품다. </li><li> 에탄올에 5 분을 품어. </li><li> 크실렌 (1:1) : 에탄올에 30 분 알을 품다. </li><li> 에탄올에 5 분을 품어. </li><li> 메탄올에서 5 분 알을 품다. </li><li> 2.5 %의 포름 알데히드와 보완, 메탄올, PBT (1:1)에 15 분 알을 품다. </li><li> PBT에서 2 × 10 분을 씻어. 이 단계에서, 슬라이드는 4 ℃에서 하룻밤 동안 유지 될 수있다 </li></ol></li><li> 세포벽 분해 : <ol><li> 얼음 세포벽 분해 믹스의 분취 량을 해동. </li><li> 종이 타월에 수직으로 배치하여 액체의 과잉 배출, 코 플린 항아리에서 슬라이드를 타고. </li><li> 아크릴 아미드 패드에 세포벽 분해 혼합 100 μl를 추가하고 23 X 46mm의 coverslip을 커버. 다른 슬라이드에 대해 반복합니다. (자료 참조) 습기 챔버에서 37 ° C에서 2 시간 동안 배양한다. </li><li> PBT에서 슬라이드 2 × 5 분을 씻으십시오. </li></ol></li><li> 치료의 RNase : <ol><li> 코 플린 항아리에서 슬라이드를 타고, 일 드레인이전과 액체의 전자 과잉. </li><li> 1 % 트윈 20 1 시간 동안 37 ° C에서 습기 챔버와 PBS에서 100 ㎍ / ml로 RNAseA 100 ㎕ 씩 각 슬라이드를 품어. </li><li> PBT에서 2 × 5 분 동안 슬라이드를 씻으십시오. </li></ol></li><li> 후 고정 및 permeabilization : <ol><li> 갓 만든 PBT-F에서 20 분 동안 후 고정합니다. </li><li> PBT에서 10 분 동안 슬라이드를 씻어. </li><li> 4 ℃에서 2 % 트윈 (20)와 PBS에서 2 시간 동안 투과 </li><li> PBT에서 2 × 5 분 동안 슬라이드를 씻어. </li></ol></li></ol> 4. 면역 염색 NOTE :이 단계에서, 일차 항체의 최적 농도는 서로 다른 희석 항체 (1:200, 1:500, 1:1000)를 사용하여 시험해야한다. <ol><li> 4 ℃에서 12 ~ 24 시간 동안 0.2 % 트윈 (20)와 PBS에 희석 차 항체 100 ㎕ 씩 각 슬라이드를 품어 </li><li> 부드러운 흔들림에 따라 실온에서 2-4 시간 동안 PBT에서 슬라이드를 씻으십시오. </li><li> 적용이차 항체 1:200 PBS + 0.2 % 트윈 20 ~ 24 시간 동안 4 ℃에서 </li><li> 부드러운 흔들림에 따라 실온에서 1 시간 동안 PBT에서 슬라이드를 씻으십시오. </li><li> 15 분 동안 PBS에서 10 ㎍ / ㎖의 프로피 디움 요오드화물로 Counterstain 후 실온에서 부드럽게 진탕 PBS에서 15 분간 린스. </li><li> 반대로 퇴색 액체의 mountant 마운트는 10 ㎍ / ㎖의 프로피 디움 요오드 보충. 설치 매체가 CLSM으로 이미지를 획득하기 전에 1 시간 동안 강화하자. </li></ol> 5. 정량 이미징 <ol><li> 이미지 수집 : <ol><li> 이상적으로 더 장시간 촬상 23 이상의 형광 신호의 보존 및 63X 글리세롤 침지 렌즈를 허용 공진 스캐닝 모드를 이용하여, CLSM을 이용한 고해상도 이미지를 획득. </li><li> 엄격히 걸쳐 따르도록 표준 획득 절차를 정의하기 위해 이러한 실험의 시작에서 레이저 강도, 게인, 핀홀, 복셀의 크기 및 줌 배율 등 획득 파라미터를 테스트일관성있는 정량적 측정을위한 모든 슬라이드. </li><li> 형광 색소 사이의 크로스 토크 (cross-talk)이 없음을 확인합니다. 존재하는 경우, 순차적 스캔을 설정합니다. 따로 따로가 아니라 동시에 전송 이미지를 획득. </li><li> x 및 y 차원에서 가능한 가장 높은 해상도와 Z 치수 (나이 퀴 스트의 규칙)의 2 배 오버 샘플링과 시리얼, 입체 영상 획득을 수행합니다. </li></ol></li><li> 이미지 처리 : <ol><li> 상업용 또는 오픈 소스 소프트웨어를 사용하여 세 가지 차원에서 일련의 이미지를 재구성. </li><li> 3D에 대한 관심의 각 핵 (또는 셀)의 주위에 윤곽 표면을 정의합니다. </li><li> 각 개체의 픽셀 강도의 합으로 각 채널에 형광의 양을. </li><li> 통계 분석을위한 데이터를 Excel로 내 보냅니다. 예를 들면 DNA 염색 신호에 대한 항체 신호를 정상화. </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

꽃 피는 식물에서 여성의 생식 계통 따라서 기술적으로 도전적인 전체 마운트의 세포 학적 염색법을 렌더링, nucellus와 난자 teguments를 포함한 여러 세포 층에 의해 둘러싸여 있습니다. 여기에서 우리는 전체 마운트의 현장 하이브리드의 면역 염색, DNA 염색 및 형광으로 세포 학적 염색에 적합한 난자의 많은 수의 준비 및 처리를 가능하게하는 효율적인 프로토콜을 제시한다. 우리는 성공적 <...

꽃 식물의 생식 계통의 설립은 SMCS의 차별화, 여성 MMC 및 남성 microspore 어머니 세포로 시작합니다. MMC는 난자 primordium의 원심 끝에서 하위 표피 nucellar 세포에서 개발하고 microspore 어머니 세포 깊은 꽃 기관 (1)의 내부에 위치하는 꽃밥 locule에 sporogenous 조직에서 개발하고 있습니다. SMCS는 유사 분열시 배우체로 야기 반수체 포자를 생산하는 세포의 감수 분열을 받다. 여성 배우체, 또는 배아 골목, 하나의 난자, 하나의 중앙 세포, 두 synergids 세 antipodals으로 구성되어 있습니다. 남성 배우체, 또는 꽃가루는, 하나의 식물 세포와 두 개의 정자 세포로 구성되어 있습니다. 남성 배우체가 상대적으로 접근 객체의 학습 남아있는 동안, 여성 배우체는 자체가 꽃 심피 묶인 난자 내부에 포함하고, 따라서 분자 및 세포 학적 분석에 대한 특정 문제를 제기한다. 그러나 최근에는 레이저를 이용한미세 절제는 transcriptomic는 MMC와 여성 gametophytic 세포 2-4의 분석을 허용하는 훌륭한 솔루션을 제공했다. 후보 유전자 발현뿐만 아니라 세포 학적 분석은 특정 직접 세포의 염색 또는 간접적으로 면역 염색을 사용하여 내인성 세포 구성 요소의 역학을 조사, 현장 하이브리드 또는 리포터 유전자 분석의 예 RNA를 허용하여, 분석한다. 특히, 함께 염색질 수정이나 염색질 구성 요소의 면역 염색으로, 생선, DNA 염색을 이용하여 세포 유전 학적 염색은 애기 장대 5 염색질 역학과 핵 조직을 명료하게 중심 접근 방식이다. 일반적으로 세포의 감수 분열이 아니라 6,7 meiocytes 식물 남성에서 조사 된 특정 염색체의 역 동성을 수반한다; 가능성이 동적 성적인 프로그래밍을 반영하는 더 큰 규모의 셀 특정 염색질 재구성, 꽃가루 개발 8 ~ 10시에 설명되어 있습니다. 대조적으로 인해여성 meiocyte와 배우체의 상대적 어려움에,이 조사는 적용 할 기술적으로 어려운 남아 있고, 자주 (아래 참조) 단면 또는 수동 해부 및 소화 효소를 필요로한다. 또, 전체의 실장 광학 명확성 널리 부족 그대로 난자의 생식 세포의 고해상도 영상에 장애물이다. 전체 마운트 난자의 염색체 조직의 세포 학적 분석을위한 고전적인 방법은 Feulgen의 염색 11 ~ 13을 사용합니다. 이는 단백질 변성을 초래하고, 따라서 염색질 구조의 파괴를 일으키는 DNA의 (차아 염소산을 이용하여) 산 가수 분해를 수반한다. 또한, 여성 meiocytes 및 gametophytic 세포의 염색체 조직 (예를 들어 14 ~ 18 참조) 세미 얇은 부분 또는 해부 배아 주머니와 MMC에 DAPI 염색과 면역 염색을 이용하여 관찰 할 수있다. 분명히, 그러나, 수동 해부 및 절편은 노동 집약적이 될 수 있습니다염색질 에피토프의 다수의 정성 및 정량 분석​​을 방해한다. 여기에서 우리는 전체 마운트 하류 세포 학적 염색의 다양한 적합한 애기 난자의 큰 숫자를 준비하는 효율적인 프로토콜을 제공합니다. 간단히, 꽃 봉오리가 정착 용액에 배양, 난자의 행은 심피에서 해부하고 꽃가루 meiocytes (19, 20)에 대해 수행으로 슬라이드에 아크릴 아미드에 포함. 내장 된 난자는 더 메탄올, 에탄올, 세포 벽 소화 permeabilization 전에 자일 렌에서 지워 고정되어 있습니다. 이 단계의 가능한 변화는 설명합니다. 샘플은 DNA 염색, 면역 염색, 물고기를 사용할 수 있습니다. 준비 모드는 효율적이며 (최대 16 슬라이드를 다른 다운 스트림 분석을 위해 하루에 제조 할 수있다) 병렬 실험 장치 할 수 있습니다. 설명 치료, 전체 마운트에 균일 한 신호를 활성화하고 잘 조직 학적, 세포의 보존세포 유형 간의 질적 및 양적 비교에 도움이 생식 세포와 주변 nucellar 세포에서 핵 조직. 보정, 3 차원 재구성 다음 CLSM 기반의 고해상도 영상은 형광 신호의 의미있는 정량적 인 측정이 가능합니다. 우리는 성공적으로 차별화 된 MMC (21)과 여성 배우체 22 개발에 염색질의 역학을 분석하기 위해이 절차를 사용; 우리는 여기에서 전체 마운트 난자의 이질 염색질 분석, 크로 마틴 면역 염색, GFP의 면역 염색과 물고기의 대표적인 결과를 제시한다. 우리는 또한 우리의 프로토콜이 다른 식물 조직 및 종에 적합 할 것이라고 믿는다.

<table border="0" cellpadding="0" cellspacing="0" width="993">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;"><a name="RANGE!A1:D39">Name of Material/ Equipment</a></td>
			<td style="width:135px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:481px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Solutions</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">BVO Fixation Buffer (based on32)</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td style="width:481px;">2mM EGTA, pH7.5, 1% (v/v) Formaldehyde,10% DMSO, 1&#215; PBS, 0.1% Tween-20</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBT</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td style="width:481px;">1&#215; PBS, 0.1% Tween-20</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBT-F</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td style="width:481px;">1&#215; PBT, 2.5% (v/v) formaldehyde</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">30% acrylamide:bisacrylamide&#160;</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td style="width:481px;">3g acrylamide, 0.33g bisacrylamide, 1&#215; PBS (prepare 10ml, store at 4&#176;C)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">200mL&#160; 5% acrylamide mix in PBS&#160;</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td style="width:481px;">34mL 30% acrylamide:bisacrylamide, 166mL 1&#215; PBS (make fresh from 30% stock)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">20% ammoniumpersulfte&#160;</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td style="width:481px;">0.2g ammoniumpersulfte, 1mL sterile water (prepare aliquots with 1mL and store at -20&#176;C)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">20% sodium sulfite&#160;</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td style="width:481px;">0.2g sodium sulfite, 1mL sterile water (prepare aliquots with 1mL and store at -20&#176;C)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell wall enzyme mix</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td style="width:481px;">0.5% (w/v) cellulase, 1% (w/v) driselase, 0.5% (w/v) pectolyase</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;"></td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reagents and Materials</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Formaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>F1635</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMSO</td>
			<td>Sigma</td>
			<td>D5879</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris</td>
			<td>Amaresco</td>
			<td>0497</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol</td>
			<td>Schaurlau</td>
			<td>ET00102500</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methanol</td>
			<td>Schaurlau</td>
			<td>ME03062500</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Xylene</td>
			<td>ROTH</td>
			<td>4436.1</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cellulase</td>
			<td>Sigma</td>
			<td>1794</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Driselase</td>
			<td>Sigma</td>
			<td>D8037</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pectolyase</td>
			<td>Sigma</td>
			<td>P5936</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween-20</td>
			<td>Merck</td>
			<td>8.22184.0500</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EGTA</td>
			<td>Sigma</td>
			<td>E-4378</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">acrylamide</td>
			<td>Sigma</td>
			<td>A-3553</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">bisacrylamide</td>
			<td>Sigma</td>
			<td>M2022</td>
			<td style="width:481px;">toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ammoniumpersulfate</td>
			<td>Sigma</td>
			<td>A9164</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium sulfite</td>
			<td>Fluka</td>
			<td>71988</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-trimethyl-Histone H3 (Lys4)</td>
			<td>Upstate</td>
			<td>07-473</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti- monomethyl-Histone H3 (Lys27)</td>
			<td>Upstate</td>
			<td>07-448</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 488~goat ~anti ~rabbit (H+L)</td>
			<td>Molecular Probe</td>
			<td>A11008</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ProlongGold</td>
			<td>Invitrogen</td>
			<td>P36934</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Propidium iodide</td>
			<td>Sigma</td>
			<td>P4170</td>
			<td style="width:481px;">toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DAPI</td>
			<td>Sigma</td>
			<td>D9542</td>
			<td style="width:481px;">toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNAse A</td>
			<td>Roche</td>
			<td>10109169001</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Coplin jar</td>
			<td>Huber &#38; CO</td>
			<td>10.055</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forceps</td>
			<td>DUMONT BIOLOGY</td>
			<td style="width:119px;"></td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Shaker</td>
			<td>Heidolph</td>
			<td>543-12310-00-0</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">Moist chamber</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td style="width:481px;">A plastic box with damp paper towel inside, a plastic support is put into the box for supporting the slides and keep slides from the water.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Superfrost Plus slide</td>
			<td>Thermo Fisher</td>
			<td>J1800AMNZ</td>
			<td style="width:481px;">Menzel-Gl&#228;ser</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FISH Tag DNA KIt</td>
			<td>Invitrogen</td>
			<td>F32947</td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">GFP booster</td>
			<td style="width:135px;">Chromotek</td>
			<td style="width:119px;"></td>
			<td style="width:481px;"></td>
		</tr>
	</tbody>
</table>

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

꽃 피는 식물에서 체세포 - 투 - 생식 세포의 운명 전환은 성인 식물의 꽃 장기에 포자 어머니 세포의 사양 (SMCS)로 표시됩니다. 여성 SMC (megaspore 어머니 세포, MMC)는 난자 primordium의 차별화 및 감수 분열을 겪는다. 선택된 반수체 megaspore는 함께 액세서리 세포, 생식 세포에 상승을 줄 것이다 다세포 여성 배우체, 난자와 중앙 셀을 형성하기 위해 유사 분열을 겪는다. 난자 내부의 MMC, meiocyte과 여성 배우체의 제한된 접근성은 세포학에 대한 기술적 도전과 세포 유전은 단일 세포 수준에서 분석한다. 특히, 세포 핵 항원의 직접 또는 간접 면역이 식물 세포 및 단일 세포 이미징 내부의 시약의 가난한 침투에 의해 손상은 전체 마운트 조직에 광학 선명도의 부족에 의해 demised된다. 따라서, 우리는 nucl을 분석 할 수있는 효율적인 방법을 개발전체 마운트 포함 된 애기 장대의 난자에 하나의 셀의 높은 해상도에서 귀 조직과 염색질 수정. 이것은 현미경 슬라이드에 아크릴 아마이드 겔의 박층의 박리 및 고정 난자의 매립에 기초한다. 내장 된 난자는 면역 염색 시약으로 조직의 명확성과 침투성을 향상을 목표로 화학 및 효소 처리를 실시하고 있습니다. 그 치료는 세포 및 염색질 조직, DNA와 단백질 항원을 유지합니다. 샘플은 염색질 면역 염색, 원위치 혼성화 (FISH) 형광 및 이질 분석을위한 DNA 염색 등 다른 하류 세포 학적 분석을 위해 사용될 수있다. 3D 재구성 다음에 높은 해상도를 가진 공 초점 레이저 주사 현미경 (CLSM) 영상은, 단일 셀 해상도에서 정량 측정 할 수 있습니다.

우리는 전체 마운트의 세포 학적 염색에 적합한 애기 난자의 대규모 준비 및 처리를위한 강력한 프로토콜을 제공합니다. 매립 덕분에, 난자는 3 차원 구조 (그림 3)를 유지한다. 또한, 광학 설명을 포함하여 조직 처리는 높은 해상도로 영상 세포 내 구조를 가능하게한다. 4 이질 염색질이 (더 디컨 볼 루션이 그림에 사용되지 않았다)도 눈에 초점을 정의, 밝은 표시 ?...

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

우리는 면역 염색, 형광 제자리 교잡, 전체 마운트 애기 장대 난자의 양적, 고해상도 이미징 다음 DNA 염색 여기를 효율적이고 신뢰할 수있는 프로토콜을 제공합니다. 이 방법은 성공적 염색질 변형 및 핵 구조를 분석하는데 사용 하였다.

전체 마운트 난자에있는 염색질 수정 및 핵 구조의 정량, 단일 세포 분석을위한 효율적인 방법<em&gt; 애기 장대</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 ...

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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 조회수.  University of Zürich.  우리는 면역 염색, 형광 제자리 교잡, 전체 마운트 애기 장대 난자의 양적, 고해상도 이미징 다음 DNA 염색 여기를 효율적이고 신뢰할 수있는 프로토콜을 제공합니다. 이 방법은 성공적 염색질 변형 및 핵 구조를 분석하는데 사용 하였다. 

비디오: 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.

조기 칙 태아의 현장 하이브리드화에서 더블 전체 산

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

연구

생물학

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.

칙의 전체 마운트 항체 스테 이닝을위한 방법

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.

마우스 콘 Photoreceptors에서 단일 셀 석션 레코딩

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.

형광을 사용하여 mRNA의 분자의 시각화 및 분석<em&gt; 제자리에서</em에서&gt; 하이브리드<em&gt; 사카 cerevisiae의</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.

측정 공간적 칼슘<sup&gt; 2 +</sup애기 장대 식물에서&gt; 신호

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.

전체 산<em&gt; 현장에서</em복족류 연체 동물 용&gt; 하이브리드 방법<em&gt; Lymnaea의 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 프로토콜 전체 마운트 피부 준비 단일 셀 해상도에서 단백질 발현을 시각화합니다

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.

전체-마운트 얼룩을 사용 하 여 3D 백색 지방 조직 구조의 시각화

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.

애기장대 및 기타 식물의 흰가루병 감염 표현형을 정확하게 평가하기 위한 쉽고 유연한 접종 방법

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.