이 작품은 Generalitat 드 Catalunya (2005 - SGR00437)에 의해 지원되었다. 저자는 그들의 자신의 기술 지원에 대한 감독과 격려, 그리고 J. 루카스에 대한 J. 블랑코와 F. 비달에 감사드립니다.

I. 샘플 처리 및 세포 고정 <ol><li> 액화까지 20 분 실온에서 멸균 용기에 정액 샘플을 둡니다. </li><li> 원심 분리기 튜브에 샘플을 전송하고 5 분 1천그램에 그것을 돌린다. </li><li> 부드럽게 파스퇴르 피펫을 사용하여 표면에 뜨는를 제거하고 폐기. </li><li> hypotonic 솔루션을 추가 (KCl, 0.075M) 미리 가열 10 ML의 최종 볼륨을 얻기 위해 소용돌이에 혼합하는 동안, 37 ° C 드롭에 의해 드롭. </li><li> 37 물 목욕에 튜브를 삽입 ° C를 30 분. </li><li> 5 분 1천g에서 원심 분리기. 조심스럽게 펠렛을 방해하지 않고, decantation로 뜨는 폐기하십시오. </li><li> 8 ML의 최종 볼륨을 얻기 위해 소용돌이에 혼합하는 동안 드롭하여 드롭 : 펠렛을 resuspending 후, 갓 Carnoy의 정착액을 (초산 3시 1분 메탄올) 준비 추가할 수 있습니다. </li><li> 흰색 펠렛을 얻기 위해 필요한만큼 여러 번 같은 점 6과 7을 반복합니다. </li><li> 신선한 메탄올 추가 : 초산 (3시 1분) (작은 펠릿 분들의 경우 확산 좋은 세포를 얻는 데 필요한 세포 농도 최종 음량을 조절하기 위해 드롭하여 드롭을 몇 방울이 높은 양의 샘플 반면에 충분합니다 정자가 회복의 최종 볼륨) 1-2 ML에 도달할 수 있습니다. 테스트 슬라이드 ungreased 슬라이드에 resuspended 샘플을 포기하고 위상 콘트라스트 현미경을 조사하여 만들 수 있습니다. 이것은 얻은 세포 분산을 확인하고 필요한 경우 추가로 슬라이드에, 그것을 정정 허용됩니다 드릴 것입니다. </li><li> 확장, 40cm의 최소 높이에서 드롭 unfrosted 슬라이드의 중앙에 세포 현탁액의 두 개 또는 세 방울 (이전에 그들을 degrease -20 ° C에서 메탄올에 저장).하려면 프로토콜을 통해 시료의 위치를​​ 사용하려면, 다이아몬드 연필을 사용하여 슬라이드의 반대편에있는 정자 확장을 포함하고있는 영역을 표시합니다. </li><li> 최소한 24 시간 동안 -20 ° C에서 슬라이드를 저장합니다. 참고 : 메탄올의 추가 : 초산 (3시 1분) 드롭 들러 혼합은 정자 집계의 형성을 피하기 위해 매우 중요한 단계 동안. </li></ol> II. Decondensation <ol><li> 슬라이드는 -20 ° C에 저장된는 (상온에서 그들을두고) 프로토콜과 함께 계속해서 사인을 알아낼 수 없을해야합니다. </li><li> 삼분 각 2X 호수 - 나트륨 구연 산염 솔루션 (2xSSC)로 연속 2 코플린 단지에있는 슬라이드를 놓습니다. </li><li> 각 코플린 병 2 분 에탄올 세척 일련의를 통해 슬라이드를 전송합니다. , 70 % 에탄올로 시작 90%에 의해 따라 100 % 완료. 실온에서 그것을두고 슬라이드를 건조시킵니다. </li><li> 37 dithiothreitol 솔루션 슬라이드 (1,4 - dithiothreitol 5mM, 1 % 트리톤 X - 100, 2 - 아미노 -2 - (히드록시 메틸) -1,3 - propanediol 50mM)을 품어 ° 인큐베이터에서 C는 염색질을 decondense 수 있습니다. 이 제품은 마리의 정자 수야 핵에 protamines의 이황화 다리 해당 코일 DNA를 깨는하여 역할을합니다. 정자의 핵에있는 DNA가 높은 압축되기 때문에 이것은 매우 중요한 단계입니다. dithiothreitol 솔루션 (DTT)의 슬라이드의 배양 시간은이 제품에 대한 샘플의 반응에 따라 조정해야합니다. 그 2로 15 분 거리에 따라 다를 수 있지만 일반적으로,이 시간은 8 분 정도입니다. </li><li> 즉시 각 코플린 3 분 2xSSC 두 개의 연속 코플린의 단지로 슬라이드를 전송합니다. </li><li> 각 코플린 병 (70 %, 90 % 및 100 % 에탄올) 2 분 에탄올 세척 일련의를 통해 슬라이드를 삽입하여 계속합니다. 상온에서 밖으로 건조에 슬라이드를하자. 참고 : 너무 짧은 노출이 어떤 신호의 부족을 초래 하리라는 반면, DTT에 과도한 노출은 프로 시저의 끝에 물고기 신호를 분산 나타날 수 있습니다. </li></ol> III. 이종 교잡 <ol><li> 73에서 포름 아미드 용액 (70 % Formamide/2xSSC) · 5 분 C와 코플린 병에 슬라이드를 잠복기하여 정자의 DNA를 변성. </li><li> 코플린 병 당 일분 (70 % 에탄올과 85 %를 시작하고 100 % 완료)에 대한 에탄올의 솔루션을 통해 슬라이드를 전송합니다. 상온에서 밖으로 건조하는 슬라이드를 둡니다. </li><li> 의 직접 5리터 추가 대응 준비 - 사용 15x15 커버 슬립 (AneuVysion 분석 멀티 컬러 프로브 패널 : CEP 18/X/Y 또는 LSI 21분의 13)에 프로브 혼합합니다. </li><li> 그것이 동영상에 나와있는, 대상 지역 및 프로브 혼합물을 함께 넣어 커버 슬립에 슬라이드를 조심스럽게 넣습니다. 고무 시멘트로 밀봉합니다. </li><li> 미리 예열 37에 슬라이드를 삽입 ° C 하이브리드화 챔버 (HYBrite ™) 및 6~24시간에 대한 ° C 37를 품어. 참고 : 정자 샘플의 변성이 필수적인 반면, 프로브를 denaturing의 필요성은 제조 업체에 의해 결정됩니다. 이 프로토콜에 사용되는 프로브 혼합물은 사용할 수 있으며,이 경우, 프로브 변성이 필요하지 않습니다. 프로브 혼합물의 볼륨 hybridized 영역의 크기에 따라 달라집니다. </li></ol> IV. 씻는다포스트 하이브 리다이 제이션 <ol><li> 하이브 리다이 제이션 챔버에서 슬라이드를 꺼내주세요. </li><li> 고무 시멘트를 제거하고 조심스럽게쪽으로 부드럽게 슬라이딩 커버 슬립을 빼낸다. </li><li> unspecific 하이브리드화 신호를 제거하려면 0.4xSSC/0.3 %와 코플린 병 2 분 동안 슬라이드를 장소 NP - 40 미리 예열 73 °에서 물을 욕조에 C. </li><li> 1 분 상온에서 2xSSC/0.1 % NP - 40 세척 솔루션에 슬라이드를 전송합니다. 상온에서 밖으로 건조에 슬라이드를하자. </li><li> 대상 지역 (18x18 커버 슬립 8 L)에 counterstaining 제품을 추가합니다. 사용되는 가장 일반적인 제품 4 &#39;, 6 diamidino - 2 - phenylindole (DAPI, Vysis 주식 회사)입니다. </li><li> 그것이 동영상에 나와있는, 커버 슬립 (커버 슬립이 손톱 바니와 함께 날인 수있는 하이브 리다이 제이션을 유지하기 위해)와 hybridized 지역을 커버. </li><li> 그들의 전망 분석까지 어둠 속에 -20 ° C에서 hybridized 슬라이드를 저장합니다. 이러한 조건 아래 슬라이드는 형광 신호 강도에 상당한 손실없이 12 개월 동안 저장할 수 있습니다. 참고 : 반대 unspecific 프로브 hybridizations을 씻지 않았 반면, 높은 온도 또는 과도한 세척 시간이 일부 신호의 제거에 발생할 수 있습니다. 추가 counterstaining 제품의 볼륨 충당하기 위해 하이브 리다이 제이션 영역의 크기에 따라 달라집니다. </li></ol> V. 시각화 준비는 DAPI / 텍사스 레드 / FITC와 단일 밴드 아쿠아, FITC와 텍사스 레드에 대한 필터 트리플 밴드 필터가 장착된 epifluorescence 현미경으로 평가할 수 있습니다. 표준 평가 기준은 정자의 핵 1의 올바른 평가를 따라야합니다. 염색체 X, Y 및 18 탐사선과 hybridized 준비 이러한 세 염색체에 대한 신호를 표시해야합니다. 모든 정상 마리의 정자 수야 한 파란 신호 (염색체 18에 해당), 그리고 녹색 신호 (X 베어링 마리의 정자 수야) 또는 빨간 신호 (Y - 베어링 마리의 정자 수야)를 제시해야합니다. 염색체 13, 21 염색체 13 염색체 21 붉은 신호 녹색 신호 프로브와 함께 hybridized 준비의 모든 정상 마리의 정자 수야에서 구별해야합니다.

<ol>
	<li>Blanco, J., Egozcue, J., Vidal, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incidence+of+chromosome+21+disomy+in+human+spermatozoa+as+determined+by+fluorescent+in-situ+hybridization.">Incidence of chromosome 21 disomy in human spermatozoa as determined by fluorescent in-situ hybridization.</a> Hum. Reprod. 11, 722-726 (1996).</li></ol>

The authors have nothing to disclose.

이 프로토콜은 정자 물고기 준비를위한 인간의 정액 샘플을 처리하는 방법에 대해 설명합니다. 이 프로토콜을 사용하면 남성의 염색체 이상 gametes를 분석하는 것이 가능합니다. 마리의 정자 수야 aneuploidy 심사는 기초 연구의 맥락과 불임 남성에게 주어진 생식 조언에 두 응용 프로그램이 있습니다. 임상 진단에 가장 널리 연구 염색체는 X, Y, 13, 18 및 21 염색체와 loci 광범위한 다른 상용 프로브은 사용할 수 있으며, 이러한 실험에서도 사용할 수 있지만.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>1,4-Dithiothreitol (DTT)</td>
<td>Material</td>
<td>Roche</td>
<td>10197777001</td>
<td>&nbsp;</td>
<tr>
<td>2-Amino-2-(hydroxymethyl)-1,3-propanediol</td>
<td>Material</td>
<td>Roche</td>
<td>10708976001</td>
<td>&nbsp;</td>
<tr>
<td>Acetic acid</td>
<td>Material</td>
<td>Merck</td>
<td>100.063</td>
<td>&nbsp;</td>
<tr>
<td>AneuVysion&reg; Multicolor DNA Probe Kit<ul style="padding:2px;"><li>20xSaline-Sodium Citrate</li><li>4,6-diamidino-2-phenylindole (DAPI II)</li><li>CEP 18/X/Y DNA probe</li><li>NP-40</li><li>LSI 13/21 DNA probe</li></ul></td>
<td>Material</td>
<td>Vysis Inc.</td>
<td>32-161075 <ul style="padding:2px;"><li>30-805850</li><li>30-804941 </li><li>30-171077</li><li>30-804820</li><li>30-171078</li></ul></td>
<td>&nbsp;</td>
<tr>
<td>Centrifuge 5804R</td>
<td>Tool</td>
<td>Eppendorf</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Centrifuge tube</td>
<td>Material</td>
<td>Nunc</td>
<td>347856</td>
<td>&nbsp;</td>
<tr>
<td>Coplin jar (glass)</td>
<td>Material</td>
<td>Barloworld Scientific</td>
<td>Hellendahl (ZCT278)</td>
<td>&nbsp;</td>
<tr>
<td>Coplin jar (plastic)</td>
<td>Material</td>
<td>Deltalab</td>
<td>191087</td>
<td>&nbsp;</td>
<tr>
<td>Cover slips (15x15)</td>
<td>Material</td>
<td>Knittel gl&auml;ser</td>
<td>4600115</td>
<td>&nbsp;</td>
<tr>
<td>Cover slips (18x18)</td>
<td>Material</td>
<td>Knittel gl&auml;ser</td>
<td>4600118</td>
<td>&nbsp;</td>
<tr>
<td>Diamond pencil</td>
<td>Material</td>
<td>Hammacher</td>
<td>335117010</td>
<td>&nbsp;</td>
<tr>
<td>Ethanol</td>
<td>Material</td>
<td>Merck</td>
<td>100.983</td>
<td>&nbsp;</td>
<tr>
<td>Formamide</td>
<td>Material</td>
<td>Roche</td>
<td>11814320001</td>
<td>&nbsp;</td>
<tr>
<td>Freezer</td>
<td>Tool</td>
<td>Zanussi</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Gloves</td>
<td>Material</td>
<td>Sanyc</td>
<td>101/3300</td>
<td>&nbsp;</td>
<tr>
<td>HYBrite&trade;</td>
<td>Tool</td>
<td>Vysis Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Immersion Oil</td>
<td>Material</td>
<td>Olympus</td>
<td>35505</td>
<td>&nbsp;</td>
<tr>
<td>Incubator</td>
<td>Tool</td>
<td>Heraeus</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Methanol</td>
<td>Material</td>
<td>Merck</td>
<td>106.009</td>
<td>&nbsp;</td>
<tr>
<td>Micropipette</td>
<td>Material</td>
<td>Labsystems</td>
<td>Finnpipette (4500000)</td>
<td>&nbsp;</td>
<tr>
<td>Micropipette replacement tip</td>
<td>Material</td>
<td>Daslab</td>
<td>16-2001</td>
<td>&nbsp;</td>
<tr>
<td>Nail varnish</td>
<td>Material</td>
<td>Quo-cosmetics</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Olympus BX60 epifluorescence microscope</td>
<td>Tool</td>
<td>Olympus</td>
<td>&nbsp;</td>
<td>Equipped with a triple-band filter for DAPI/Texas Red/FITC and single-band filters for Aqua, FITC and Texas Red.</td>
</tr>
<tr>
<td>Pasteur Pipette</td>
<td>Material</td>
<td>Rubilabor</td>
<td>211.0230</td>
<td>&nbsp;</td>
<tr>
<td>Phase contrast microscope</td>
<td>Tool</td>
<td>Nikon Diaphot</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Plastic pipette (3ml)</td>
<td>Material</td>
<td>Deltalab</td>
<td>200007</td>
<td>&nbsp;</td>
<tr>
<td>Potassium chloride (KCl)</td>
<td>Material</td>
<td>Fluka</td>
<td>60128</td>
<td>&nbsp;</td>
<tr>
<td>Rubber cement</td>
<td>Material</td>
<td>Best-test</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Slides</td>
<td>Material</td>
<td>Knittel gl&auml;ser</td>
<td>4520022</td>
<td>&nbsp;</td>
<tr>
<td>Slide Saver Boxes</td>
<td>Material</td>
<td>Deltalab</td>
<td>19276.1</td>
<td>&nbsp;</td>
<tr>
<td>Sterile container</td>
<td>Material</td>
<td>Deltalab</td>
<td>409726</td>
<td>&nbsp;</td>
<tr>
<td>Syringe</td>
<td>Material</td>
<td>PentaFerte</td>
<td>002022300</td>
<td>&nbsp;</td>
<tr>
<td>Thermometer</td>
<td>Material</td>
<td>Comark Instruments, Inc</td>
<td>KM12</td>
<td>&nbsp;</td>
<tr>
<td>Tri-distilled water</td>
<td>Material</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>Material</td>
<td>Sigma</td>
<td>9002-93-1</td>
<td>&nbsp;</td>
<tr>
<td>Tweezers</td>
<td>Material</td>
<td>B/Braun</td>
<td>Aesculap (BD224R)</td>
<td>&nbsp;</td>
<tr>
<td>Vertical laminar flow hood</td>
<td>Tool</td>
<td>Burdinola</td>
<td>OR-ST 1500</td>
<td>&nbsp;</td>
<tr>
<td>Vortex mixer</td>
<td>Tool</td>
<td>Fisher Scientific</td>
<td>FB15012</td>
<td>&nbsp;</td>
<tr>
<td>Water bath</td>
<td>Tool</td>
<td>Raypa&reg;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

fluorescence in situ hybridization (fish) protocol in human sperm

Aneuploidies은 인간에서 가장 자주 염색체 이상입니다. 이러한 이상의 대부분은 부모의 gametogenic 과정 meiotic 오류로 인한. 인간의 남성에서 이러한 오류가 자손에게 이러한 이례적인 전송의 증가 위험을 나타내는 수치 염색체 이상과 마리의 정자 수야의 생산으로 이어질 수 있습니다. 이러한 이유로, 정자의 핵에 원위치 하이브리드화 (물고기)의 형광의 기술은 광범위하게 임상 진단의 맥락에 통합 프로토콜되고있다. 이러한 행위는 유전자의 생식 조언을 추구 환자의 gametes의 수치 염색체 이상의 주파수의 예측을 제공합니다. 지금까지 대부분의 자주 정자 생선 분석에 포함된 염색체는 염색체 X, Y, 13, 18과 21입니다. 이 비디오 문서에서는 어떻게 현미경의 결과를 시각화하는 방법에 정자 물고기 준비를 얻기 위해 진행 정자 염색질을 decondense 및 변성하는 방법, 인간의 정액 샘플을 처리하고 해결하는 방법을 단계적으로, 그리고 설명합니다. 가장 관련성이 높은 단계의 특별 연설은 최상의 결과를 달성하기 위해 주어집니다.

Watch this Scientific Journal Video about Fluorescence in situ hybridization FISH Protocol in Human Sperm at JoVE.com

Fluorescence in situ hybridization FISH Protocol in Human Sperm

이 비디오 기사는 양질의 형광을 달성하기 위해 정액 샘플을 처리하는 방법을 단계적으로, 설명<em&gt; 현장에서</em인간 마리의 정자 수야에&gt; 하이브 리다이 제이션. 얻은 준비는 임상 진단의 맥락에서 aneuploidy 심사 사용할 수 있습니다.

형광 현장에서</em인간의 정자에서> 하이브리드화 (물고기) 프로토콜

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic ...

fluorescence-in-situ-hybridization-fish-protocol-in-human-sperm

Nanyang Technological University

Research

JoVE Journal

Biology

Fluorescence in situ hybridization (FISH) Protocol in Human Sperm

30.0K 조회수.  Universitat Autónoma de Barcelona. 이 비디오 기사는 양질의 형광을 달성하기 위해 정액 샘플을 처리하는 방법을 단계적으로, 설명 현장에서 하이브 리다이 제이션. 얻은 준비는 임상 진단의 맥락에서 aneuploidy 심사 사용할 수 있습니다.

비디오: Fluorescence in situ hybridization FISH Protocol in Human Sperm

Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species

The high-throughput expression analysis technologies available today give scientists an overflow of expression profiles but their resolution in terms of tissue specific expression is limited because of problems in dissecting individual tissues. Expression data needs to be confirmed and complemented with expression patterns using e.g. in situ hybridization, a technique used to localize cell specific mRNA expression. The in situ hybridization method is laborious, time-consuming and often requires extensive optimization depending on species and tissue. In situ experiments are relatively more difficult to perform in woody species such as the conifer Norway spruce (Picea abies). Here we present a modified DIG in situ hybridization protocol, which is fast and applicable on a wide range of plant species including P. abies. With just a few adjustments, including altered RNase treatment and proteinase K concentration, we could use the protocol to study tissue specific expression of homologous genes in male reproductive organs of one gymnosperm and two angiosperm species; P. abies, Arabidopsis thaliana and Brassica napus. The protocol worked equally well for the species and genes studied. AtAP3 and BnAP3 were observed in second and third whorl floral organs in A. thaliana and B. napus and DAL13 in microsporophylls of male cones from P. abies. For P. abies the proteinase K concentration, used to permeablize the tissues, had to be increased to 3 g/ml instead of 1 g/ml, possibly due to more compact tissues and higher levels of phenolics and polysaccharides. For all species the RNase treatment was removed due to reduced signal strength without a corresponding increase in specificity. By comparing tissue specific expression patterns of homologous genes from both flowering plants and a coniferous tree we demonstrate that the DIG in situ protocol presented here, with only minute adjustments, can be applied to a wide range of plant species. Hence, the protocol avoids both extensive species specific optimization and the laborious use of radioactively labeled probes in favor of DIG labeled probes. We have chosen to illustrate the technically demanding steps of the protocol in our film.
Anna Karlgren and Jenny Carlsson contributed equally to this study.
Corresponding authors: Anna Karlgren at Anna.Karlgren@ebc.uu.se and Jens F. Sundstr&#246;m at Jens.Sundstrom@vbsg.slu.se

Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species

We describe a modified DIG in situ hybridization protocol, which is fast and applicable on a wide range of plant species including Norway spruce. With just a few adjustments, including altered RNase treatment and proteinase K concentration, the protocol may be used in studies of different tissues and species.

비 - 방사성 현장에서 하이브 리다이 제이션 노르웨이 스프루스에 대한 해당 프로토콜과 식물 종의 범위

The high-throughput expression analysis technologies available today give scientists an overflow of expression profiles but their resolution in terms of ...

연구

생물학

Techniques for Imaging Ca2+ Signaling in Human Sperm

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca2+-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca2+-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca2+ response as % change in fluorescence is obtained.

Techniques for Imaging Ca2+ Signaling in Human Sperm

Stimulus-evoked [Ca2+]i signals of individual human sperm are assessed. Motile cells are loaded with Ca2+-sensitive fluorescent dye (AM-ester method) and immobilised in a perfusable chamber. Cells are imaged by time-lapse fluorescence microscopy and stimulated via the perfusing medium. Responses of single cells (or regions) are analysed offline using Excel.

이미징 CA의 기술 2 + 인간 정자의 시그널링

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

형광에 의한 바이러스성 RNA의 탐지<em&gt; 현장에서</em&gt; 하이브리드화 (물고기)

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 protein variants encoded by the var genes.
The haploid P. falciparum genome harbors approximately 60 different var genes of which only one has been believed to be transcribed per cell at a time during the blood stage of the infection. How such mutually exclusive regulation of var gene transcription is achieved is unclear, as is the identification of individual var genes or sub-groups of var genes associated with different receptors and the consequence of differential binding on the clinical outcome of P. falciparum infections. Recently, the mutually exclusive transcription paradigm has been called into doubt by transcription assays based on individual P. falciparum transcript identification in single infected erythrocytic cells using RNA fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE1.
Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human erythrocytes. The method is based on the use of digoxigenin- and biotin- labeled antisense RNA probes using the TSA Plus Fluorescence Palette System2 (Perkin Elmer), microscopic analyses and freshly selected P. falciparum IE. The in situ hybridization method can be used to monitor transcription and regulation of a variety of genes expressed during the different stages of the P. falciparum life cycle and is adaptable to other malaria parasite species and other organisms and cell types.

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH

Fluorescent in situ hybridization (FISH) to identify mRNA transcripts in individual cells allows analysis of polygenic activity such as the simultaneous transcription of more than one member of the var multigene family in Plasmodium falciparum infected erythrocytes 1. The technique is adaptable and can be used on different types of genes, cells and organisms.

RNA 형광하여 단일 세포 유전자 전사의 분석<em&gt; 현장에서</em&gt; 하이브리드 (물고기)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 ...

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 ...

Combined DNA-RNA Fluorescent In situ Hybridization (FISH) to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in cytogenetics and cancer diagnostics, and can detect aberrations of the genome, which often has important clinical implications. RNA FISH can be used to detect RNA molecules in cells and has provided important insights in regulation of gene expression. Combining DNA and RNA FISH within the same cell is technically challenging, as conditions suitable for DNA FISH might be too harsh for fragile, single stranded RNA molecules. We here present an easily applicable protocol which enables the combined, simultaneous detection of Xist RNA and DNA encoded by the X chromosomes. This combined DNA-RNA FISH protocol can likely be applied to other systems where both RNA and DNA need to be detected.

Combined DNA-RNA Fluorescent In situ Hybridization FISH to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) allows the detection of nucleic acids in their native environment within cells. We here describe a protocol for the combined, simultaneous detection of RNA and DNA by means of FISH, which can be used to study X chromosome inactivation in mouse embryonic stem cells.

결합 된 DNA-RNA 형광<em&gt; 현장에서</em&gt; 하이브리드 화 (물고기) 차별화 된 여성 마우스 배아 줄기 세포에서 X 염색체의 불 활성화를 연구하는

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in ...

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly effective at mapping highly repetitive sequences to heterochromatic regions, where computational approaches face prohibitive challenges. Here we describe a streamlined protocol for DNA ISH that circumvents formamide washes that are standard steps in other DNA ISH protocols. Our protocol is optimized for hybridization with short single strand DNA probes that carry fluorescent dyes, which effectively mark repetitive DNA sequences within heterochromatic chromosomal regions across a number of different insect tissue types. However, applications may be extended to use with larger probes and visualization of single copy (non-repetitive) DNA sequences. We demonstrate this method by mapping several different repetitive sequences to squashed chromosomes from Drosophila melanogaster neural cells and Nasonia vitripennis spermatocytes. We show hybridization patterns for both small, commercially synthesized probes and for a larger probe for comparison. This procedure uses simple laboratory supplies and reagents, and is ideal for investigators who have little experience with performing DNA ISH.

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

Here, we present a simple method for performing fluorescence DNA in situ hybridization (DNA ISH) to visualize repetitive heterochromatic sequences on slide-mounted chromosomes. The method requires minimal reagents and it is versatile for use with short or long probes, different tissues, and detection with fluorescence or non-fluorescence-based signals.

형광 DNA를위한 간단한 방법<em&gt; 현장에서</em&gt; 숙청 염색체에 하이브리드

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly ...

Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization (FISH) with PNA Probes in Caenorhabditis elegans

Telomere is a ribonucleoprotein structure that protects chromosomal ends from aberrant fusion and degradation. Telomere length is maintained by telomerase or an alternative pathway, known as alternative lengthening of telomeres (ALT)1. Recently, C. elegans has emerged as a multicellular model organism for the study of telomere and ALT2. Visualization of repetitive sequences in the genome is critical in understanding the biology of telomeres. While telomere length can be measured by telomere restriction fragment assay or quantitative PCR, these methods only provide the averaged telomere length. On the contrary, fluorescence in situ hybridization (FISH) can provide the information of the individual telomeres in cells. Here, we provide protocols and representative results of the method to determine telomere length of C. elegans by fluorescent in situ hybridization. This method provides a simple, but powerful, in situ procedure that does not cause noticeable damage to morphology. By using fluorescently labeled peptide nucleic acid (PNA) and digoxigenin-dUTP-labeled probe, we were able to visualize two different repetitive sequences: telomere repeats and template of ALT (TALT) in C. elegans embryos and gonads.

Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization FISH with PNA Probes in Caenorhabditis elegans

We report a concise procedure of fluorescence in situ hybridization (FISH) in the gonad and embryos of Caenorhabditis elegans for observing and quantifying repetitive sequences. We successfully observed and quantified two different repetitive sequences, telomere repeats and template of alternative lengthening of telomeres (TALT).

관측 및 텔로미어의 정량 및 반복 시퀀스 사용하여 형광<em&gt; 현장에서</emPNA 프로브에와&gt; 하이브리드 (FISH)<em&gt; 예쁜 꼬마 선충</em

Telomere is a ribonucleoprotein structure that protects chromosomal ends from aberrant fusion and degradation. Telomere length is maintained by telomerase ...

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

A method is described for labeling individual messenger RNA (mRNA) transcripts in fixed bacteria for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH allows the measurement of cell-to-cell variability in mRNA copy number of genes of interest, as well as the subcellular location of the transcripts. The main steps involved are fixation of the bacterial cell culture, permeabilization of cell membranes, and hybridization of the target transcripts with sets of commercially available short fluorescently-labeled oligonucleotide probes. smFISH can allow the imaging of the transcripts of multiple genes in the same cell, with limitations imposed by the spectral overlap between different fluorescent markers. Following completion of the protocol illustrated below, cells can be readily imaged using a microscope coupled with a camera suitable for low-intensity fluorescence. These images, together with cell contours obtained from segmentation of phase contrast frames, or from cell membrane staining, allow the calculation of the mRNA copy number distribution of a sample of cells using open-source or custom-written software. The labeling method described here can also be applied to image transcripts with stochastic optical reconstruction microscopy (STORM).

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

This manuscript describes a method for labeling individual messenger RNA (mRNA) transcripts with fluorescently-labeled DNA probes, for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH is a visualization method that allows the simultaneous detection, localization, and quantification of single mRNA molecules in fixed individual cells.

단일 분자 형광 제자리에서 교 잡 실험에 대 한 개별 대장균 세포에 성적표를 라벨에 대 한 방법

A method is described for labeling individual messenger RNA (mRNA) transcripts in fixed bacteria for use in single-molecule fluorescence in situ ...

Fish Sperm Assessment Using Software and Cooling Devices

For gamete quality evaluation, there are innovative, rapid, and quantitative techniques that can provide useful data for aquaculture. Computerized systems for sperm analysis were developed to measure several parameters and one of the most commonly measured is the sperm motility.
Initially, this computer technology was designed for mammalian species, although it can also be used for fish sperm analysis. Fish have specific features that can affect sperm assessment such as a short motility time after activation and, in some cases, adaptation to lower temperatures. Thus, it is necessary to modify both software and hardware components to make motility analysis more efficient for fish sperm analysis. For mammalian sperm, the heating plate is used to maintain optimal temperatures of spermatozoa. However, for some fish species, it is advantageous to use a lower temperature to prolong the duration of motility, since the sperm remain active for less than 2 min. Therefore, cooling devices are necessary to refrigerate samples at constant temperature over the time of analysis, including on the optical microscope. This protocol describes the analysis of fish sperm motility using software for sperm analysis and new cooling devices to optimize the results.

The present protocol describes a procedure of fish sperm assessment using computer-assisted sperm analysis and cooling devices. The software gives a rapid, accurate and quantitative analysis of fish sperm quality based on spermatozoa motility, which can be a useful tool in aquaculture to improve reproduction success.

생선 정자 평가 소프트웨어를 사용 하 여 및 냉각 장치

For gamete quality evaluation, there are innovative, rapid, and quantitative techniques that can provide useful data for aquaculture. Computerized systems ...

형광 현장에서 하이브리드화 (물고기) 프로토콜