Name: paraffin-embedded and frozen sections of drosophila adult muscles
Uploaded: 2010-12-27T17:30:00
Duration: 7 min 28 s
Description: Watch this Scientific Journal Video about Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles at JoVE.com

우리는 우리가 cryo - 마이크로톰를 사용할 수에 대한 교수 Eichele 감사합니다. 작품은 맥스 - 플랑크 - Gesselschaft에 의해 투자되었다.

1. 준비 <ol><li> 갓 3이. 각각 비례 6시 3분 1초에 절대 에탄올, 클로로포름과 빙초산을 결합하여 Carnoy의 정착액을 준비뿐만 아니라 잠수함이 잠수 체포에 대한 모든 솔루션 (추천 시약 섹션을 참조) 유리 얼룩의 단지에 보관해야합니다. </li><li> 준비 알루미늄 호일은 체포에 대한 올바른 크기를 포켓. </li><li> 2 X 40 % 에탄올, 70 % 에탄올, 2 X 100 % 에탄올, methylbenzoate (MB), 50분의 50 V / V MB의 파라핀, 2 X의 파라핀 : 단지를 얼룩에 다음과 같은 솔루션을 준비합니다. 60-65 ° C.에 보육 세트에서 MB의 파라핀 용기를 배치 </li><li> 60-65 ° C.에 호일 주머니에 붓다에게 따뜻한 파라핀 </li></ol> 2. 목걸이에 파리를 고정 <ol><li> 당신이 비행에 대한 진입 점을 볼 수있는 수직 위치에 테이프를 사용하여 양안에서 옷깃 4 연결합니다. </li><li> 마취는 얼음 블록을 사용하여 이산화탄소 또는 저체온증을 통해를 사용하여 날아. 파리를 동결하지 않도록주의하십시오. </li><li> 포셉를 사용하여 (날개와 날개 아래 복부 위에 머리와 흉부) 제대로 지향 칼라로 자신의 날개와 자리를 잡아 개별 파리 픽업. 10-20 파리 쉽게 칼라에 맞게해야합니다. </li></ol> 참고 : 여러 genotypes을 분석하는 경우, 칼라 번호와 해당 유전자형의 노트를 만들기 위해 잊지 마세요. 3. Drosophila Thoraxes의 파라핀 섹션 <ol><li> Carnoy의 솔루션에 대한 칼라를 재배치 4에서 조직을 수정 ° C를 밤이여. </li><li> 고정 후, 에탄올의 농도 증가를 사용하여 샘​​플을 탈수. 십분 40% (2 회) 70 %와 실온에서 100 % (2 회) 에탄올에 각각의 잠수함 칼라하십시오. 다음 부화의 각 30 분 MB의 메가바이트 + 파라핀 용액 (1:1)의 칼라 및 60-65시 60 분 각 파라핀의 두 가지 변경 사항 ° C.의 칼라를 침투 빠르게 호일 주머니에 칼라를 재배치하고 녹인 (60-65 ° C) 파라핀과 함께 입력합니다. 실온에서 그것을 장소와 파라핀은 (이것은 야간두고하는 것이 가장 좋은 방법입니다) 하드가 될 수 있습니다. 칼라가이 (세로 또는 가로)가 필요 섹션의 방향에 따라 서로 다른 방향에있는 호일 주머니에 놓을 수 있습니다. </li><li> 호일의 체포와 함께 건조 파라핀 블록 포장을 푸는 부드럽게 파라핀 블록에서 칼라를 구분한다. 날카로운 블레이드 또는 메스의 도움으로 조심스럽게 파리 조직 주변에서 엑스트라 파라핀을 잘라 버릴거야. </li><li> 회전 마이크로톰에서 7-10 μm의 섹션 단계로 파라핀 블록을 잘라 상처 조직이 37 ° C 물 목욕으로 평면 떠 수 있습니다. 극지 슬라이드에 sectioned 조직을 놓고 밤 동안 건조 수 있습니다. 이 슬라이드는 hematoxyline과 eosin 염색법과 함께 사용할 수 있습니다 (그림 1A - D), toluidine 파랑, 아닐린 청색 또는 기타 죽어가 조직 구조를 시각화하기 위해뿐만 아니라 항체 얼룩에 관해서는 (그림 1E - E ``). </li></ol> 4. Drosophila Thoraxes의 Cryosections <ol><li> 파라핀 섹션과 마찬가지로, 칼라와 패션 알루미늄 호일 주머니에 파리를 준비합니다. 냉동 냉각기가 샘플을 준비하는 데 필요한 것입니다. 쿨러가 -60 주위에 있는지 확인 ° C,이 온도에 도달하도록하기 위해서 약간의 에탄올과 드라이 아이스를 사용합니다. 실험을 시작하기 전에 시간을 진정하고 공기 거품의 형성을 최소화하기 위해 4 ° C 냉장고에 거꾸로 cryo - 퍼가기 매체의 병 (조직 테크 OCT 화합물)을 넣어. </li><li> 냉동 냉각기 내부에 몇 분 동안 미리 냉각 빠르게 cryo - 포함 화합물로 작성되었습니다 호일 주머니에 파리와 칼라를 변경합니다. 30-10 분 샘플 동결하자. 신중하게, 쿨러 내부에 형성된 블록 포장을 푸는 부드럽게 삽입 블록에서 칼라를 별도의 최소한 하루 -20 ° C에 넣어. </li><li> 10-15 μm의의 단면 두께 -15 및 -18 ° C 사이는 cryo - 마이크로톰에 냉동 근육을 잘라. 편광 슬라이드에 삽입하고 추가 처리를 할 준비가 때까지 -20 ° C에서 보관. 우리는 사전에 항체 염색법에 실온에서 10 분 4 %의 포름 알데히드 PBS 용액에 조직을 고정하는 것이 좋습니다. </li></ol> 5. Drosophila 근육의 Lipids 감지 지질 방울은 기름 붉은 O가 지버 및 Thummel 5 채택된 프로토콜을 사용 cryosections에 얼룩로 검색하실 수 있습니다. <ol><li> 고정 후, 3 기름 빨간색 O의 H는​​ 상온에서 얼룩을 위해 10 분 및 부화에 대한 프로필렌 글리콜에 equilibrated 5 분에 대해 두 번 물로 슬라이드를 씻으십시오. 다음 프로필렌 글리콜과 PBS에서 30 분 5 분 샘플에게 2 번 씻는다. 30 % 글리세롤에 마운트합니다. </li></ol> 6. 대표 결과 : <img src="/files/ftp_upload/2438/2438fig1.jpg" alt="그림 1" /> 무화과우레 1. Parrafin - 임베디드 섹션 간접 비행 근육의 Hematoxyline 및 eosine 스테인드 가로 (AB)와 세로 (CD) 섹션. A와 C는 정상적인 구조 근육을 보여줍니다. 크기와 형태 근육의 비정상는 각각 B와 D (검은색 화살표)에 표시됩니다. (EF) 얼룩 핵 방지 LamC, 핵 봉투 표지와 DAPI와 Drosophila의 흉부 자국의 가로 섹션을 참조하십시오. 정상의 확대 (빨간색 화살표)과 악화 (노란색 화살표) 근육 (F). G는 LamC와 DAPI와 Drosophila 창자 자국의 섹션을 나타냅니다. <img src="/files/ftp_upload/2438/2438fig2.jpg" alt="그림 2" /> 그림 2. 냉동 섹션 오일 붉은 O, 지질 방울의 라벨 Drosophila의 흉부 자국의 A. 트랜스 냉동 섹션. 간접 비행 근육의 B. 세로 냉동 섹션 방지 DG, 근육 sarcolemma 마커와 DAPI 물들다.

<ol>
	<li>Miller, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+internal+anatomy+and+histology+of+the+imago+of+Drosophila+melanogaster.">The internal anatomy and histology of the imago of Drosophila melanogaster.</a> , (1950).</li><li>Shcherbata, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+muscle+and+neuronal+disorders+in+a+Drosophila+model+of+muscular+dystrophy.">Dissecting muscle and neuronal disorders in a Drosophila model of muscular dystrophy.</a> The EMBO journal. 26, 481-481 (2007).</li><li>Kucherenko, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+modifier+screens+reveal+new+components+that+interact+with+the+Drosophila+dystroglycan-dystrophin+complex.">Genetic modifier screens reveal new components that interact with the Drosophila dystroglycan-dystrophin complex.</a> PloS one. 3, e2418-e2418 (2008).</li><li>Puchtler, H., Waldrop, F. S., Conner, H. M., Terry, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carnoy+fixation:+practical+and+theoretical+considerations.">Carnoy fixation: practical and theoretical considerations.</a> Histochemie. 16, 361-36 (1968).</li><li>Ashburner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Drosophila - A Laboratory Manual. , (1989).</li><li>Sieber, M. H., Thummel, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+DHR96+nuclear+receptor+controls+triacylglycerol+homeostasis+in+Drosophila.">The DHR96 nuclear receptor controls triacylglycerol homeostasis in Drosophila.</a> Cell metabolism. 10, 481-481 (2009).</li></ol>

관심 없음 충돌 선언하지 않습니다.

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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>Stainless steel collars</td>
<td>&nbsp;</td>
<td>self-made</td>
<td>&nbsp;</td>
<td>Specially constructed</td>
</tr>
<tr>
<td>Forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools GmbH</td>
<td>11295-10</td>
<td>&nbsp;</td>
<tr>
<td>Aluminum foil</td>
<td>&nbsp;</td>
<td>Any</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Blade or scalpel</td>
<td>&nbsp;</td>
<td>Any</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Wheaton macro staining jar</td>
<td>&nbsp;</td>
<td>Wheaton Science Products </td>
<td>900200</td>
<td>&nbsp;</td>
<tr>
<td>Microtome</td>
<td>&nbsp;</td>
<td>Carl Zeiss</td>
<td>&nbsp;</td>
<td>Model: Hyrax M25</td>
</tr>
<tr>
<td>Cryo-microtome</td>
<td>&nbsp;</td>
<td>Leica </td>
<td>&nbsp;</td>
<td>Model CM3050S</td>
</tr>
<tr>
<td>60-65&deg;C Incubator</td>
<td>&nbsp;</td>
<td>Any</td>
<td>&nbsp;</td>
<td>Large enough to hold at least 4 staining jars</td>
</tr>
<tr>
<td>Freezing cooler with metal block</td>
<td>&nbsp;</td>
<td>Any</td>
<td>&nbsp;</td>
<td>Store at -80&deg;C</td>
</tr>
<tr>
<td>Super-frost slides</td>
<td>&nbsp;</td>
<td>Thermo Fisher Scientific</td>
<td>9161155</td>
<td>&nbsp;</td>
<tr>
<td>Cover slips</td>
<td>&nbsp;</td>
<td>Any </td>
<td>&nbsp;</td>
<td>Recommend 24 X 40 mm</td>
</tr>
<tr>
<td>Chloroform</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>288306</td>
<td>Analytical grade </td>
</tr>
<tr>
<td>Glacial acetic acid</td>
<td>&nbsp;</td>
<td>Merck</td>
<td>100063</td>
<td>Analytical grade</td>
</tr>
<tr>
<td>Ethanol</td>
<td>&nbsp;</td>
<td>Merck</td>
<td>100983</td>
<td>Analytical grade</td>
</tr>
<tr>
<td>Methylbenzoate</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>M29908-500G</td>
<td>Analytical grade</td>
</tr>
<tr>
<td>Paraplast plus</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>76258</td>
<td>Paraffin</td>
</tr>
<tr>
<td>Tissue-Teck O.C.T. compound</td>
<td>&nbsp;</td>
<td>Sakura</td>
<td>4583</td>
<td>&nbsp;</td>
<tr>
<td>16% formaldehyde, methanol free</td>
<td>&nbsp;</td>
<td>Polysciences</td>
<td>18814</td>
<td>&nbsp;</td>
<tr>
<td>Glycerol</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>G5150-1L</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

paraffin-embedded and frozen sections of drosophila adult muscles

인간의 근육 dystrophies 및 myopathies의 분자 특성은 근육 질환 및 근육 사양의 유전자 분석의 복잡도를 발표했다, 모델 시스템의 형성과 기능은 근육 생리에 중요한 통찰력을 제공하고 있습니다. 따라서, 식별 및 근육 손상을 기초 분자 메커니즘을 특성화하는 것이 중요합니다. 성인 Drosophila 멀티 섬유 근육의 구조는 척추 평형으로 뻗은 줄이있는 근육 1 Drosophila의 유전자 tractability이에게 dystrophic 근육 형태를 분석하고 노화 성인에서 근육 기능에 영향을 미치는 프로세스 2 날아 성격에 큰 시스템을 만들었습니다 유사. 여기 파라핀 - 임베디드 및 Drosophila 흉부 근육의 부분을 냉동을 준비 histological 기술을 제시한다. 이러한 준비는 고전적인 histological 얼룩 물들일와 단백질 검출 염료로 표시되도록 조직을 허용하고, 특히 cryosections 그대로 근육에서 단백질의 immunohistochemical 검출에 이상적입니다. 이것은 근육 조직 구조, 형태학의 결함의 식별, 그리고 근육 / Drosophila 성인 근육의 신경 세포 특정 단백질에 대한 표현 패턴의 감지 분석을 위해 수 있습니다. 이러한 기술은 또한 약간 다른 신체 부위의 sectioning 위해 수정할 수 있습니다.

Watch this Scientific Journal Video about Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles at JoVE.com

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

근육 손상을 기본 메커니즘의 확인이 중요합니다. 여기 파라핀 - 임베디드 및 Drosophila 흉부 근육의 부분을 냉동을 준비 histological 기술을 제시한다. 이것은 근육 형태학 및 단백질 및 기타 근육 세포 구성 요소의 로컬 리 제이션의 분석이 가능합니다.

의 파라핀 - 임베디드 및 냉동 섹션 Drosophila 성인 근육

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

paraffin-embedded-and-frozen-sections-of-drosophila-adult-muscles

Research

JoVE Journal

Biology

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Whole Cell Recordings from Brain of Adult Drosophila

In this video, we demonstrate the procedure for isolating whole brains from adult Drosophila in preparation for recording from single neurons. We begin by describing the dissecting solution and capture of the adult females used in our studies. The procedure for removing the whole brain intact, including both optic lobes, is illustrated. Dissection of the overlying trachea is also shown.  The isolated brain is not only small but needs special care in handling at this stage to prevent damage to the neurons, many of which are close to the outer surface of the tissue. We show how a special holder we developed is used to stabilize the brain in the recording chamber. A standard electrophysiology set up is used for recording from single neurons or pairs of neurons. A fluorescent image, viewed through the recording microscope, from a GAL4 line driving GFP expression (GH146) illustrates how projection neurons (PNs) are identified in the live brain. A high power Nomarski image shows a view of a single neuron that is being targeted for whole cell recording. When the brain is successfully removed without damage, the majority of the neurons are spontaneously active, firing action potentials and/or exhibiting spontaneous synaptic input. This in situ preparation, in which whole cell recording of identified neurons in the whole brain can be combined with genetic and pharmacological manipulations, is a useful model for exploring cellular physiology and plasticity in the adult CNS.

This video demonstrates the procedure for isolating whole brains from adult Drosophila in preparation for recording from single neurons using standard whole cell technology. It includes images of GFP labeled cells and neurons viewed during recording.

성인 Drosophila의 머리에서 전체 셀 레코딩

In this video, we demonstrate the procedure for isolating whole brains from adult Drosophila in preparation for recording from single neurons. We begin by ...

연구

생물학

High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field. Studying locomotor behavior in Drosophila melanogaster relies on the ability to be able to quantify changes in motion during or in response to a given task. For this reason, a high-resolution video tracking system, such as the one we describe in this paper, is a valuable tool for measuring locomotion in real-time. Our protocol involves the use of an initial air pulse to break the flies momentum, followed by a thirty second filming period in a square chamber. A tracking program is then used to calculate the instantaneous speed of each fly within the chamber in 10 msec increments. Analysis software then compiles this data, and outputs a variety of parameters such as average speed, max speed, time spent in motion, acceleration, etc. This protocol will discuss proper feeding and management of flies for behavioral tasks, handling flies without anesthetization or immobilization, setting up a controlled environment, and running the assay from start to finish.

The study of complex locomotor behavior in Drosophila melanogaster is dependent upon the ability to quantify changes in a given fly's movement. This article demonstrates how to do this using a high-resolution tracking system.

성인 Drosophila Melanogaster의 로코의 고해상도 비디오 추적

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field.  Studying ...

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three preparations of the brain and visual system that cover the range from the developing eye disc-brain complex in the developing pupae to individual eye and brain dissection from adult flies. All protocols are optimized for the live culture of the preparations. However, we also present the conditions for fixed tissue immunohistochemistry where applicable. Finally, we show live imaging conditions for these preparations using conventional and resonant 4D confocal live imaging in a perfusion chamber. Together, these protocols provide a basis for live imaging on different time scales ranging from functional intracellular assays on the scale of minutes to developmental or degenerative processes on the scale of many hours.

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

This protocol describes three Drosophila preparations: 1) adult brain dissection, 2) adult retina dissection and 3) developing eye disc- brain complexes dissection. Emphasis is laid on special preparation techniques and conditions for live imaging, although all preparations can be used for fixed tissue immunohistochemistry.

개발 및 성인의 준비 Drosophila 두뇌와 라이브 영상을위한 Retinae

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three ...

Dissection of Oenocytes from Adult Drosophila melanogaster

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides protection against desiccation and other environmental challenges. Several of these cuticular hydrocarbon (CHC) compounds also function as semiochemical signals, and as such mediate pheromonal communications between members of the same species, or in some instances between different species, and influence behavior. Specialized cells referred to as oenocytes are regarded as the primary site for CHC synthesis. However, relatively little is known regarding the involvement of the oenocytes in the regulation of the biosynthetic, transport, and deposition pathways contributing to CHC output. Given the significant role that CHCs play in several aspects of insect biology, including chemical communication, desiccation resistance, and immunity, it is important to gain a greater understanding of the molecular and genetic regulation of CHC production within these specialized cells. The adult oenocytes of D. melanogaster are located within the abdominal integument, and are metamerically arrayed in ribbon-like clusters radiating along the inner cuticular surface of each abdominal segment. In this video article we demonstrate a dissection technique used for the preparation of oenocytes from adult D. melanogaster. Specifically, we provide a detailed step-by-step demonstration of (1) how to fillet prepare an adult Drosophila abdomen, (2) how to identify the oenocytes and discern them from other tissues, and (3) how to remove intact oenocyte clusters from the abdominal integument. A brief experimental illustration of how this preparation can be used to examine the expression of genes involved in hydrocarbon synthesis is included. The dissected preparation demonstrated herein will allow for the detailed molecular and genetic analysis of oenocyte function in the adult fruit fly.

Dissection of Oenocytes from Adult Drosophila melanogaster

In insects, the oenocytes produce cuticular hydrocarbon compounds. These compounds protect against desiccation and facilitate chemical communication. Here we demonstrate a dissection technique used to isolate the oenocytes from adult Drosophila melanogaster, and illustrate how this preparation can be utilized to study genes involved in hydrocarbon synthesis.

성인에서 Oenocytes의 해부 Drosophila melanogaster</em

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides ...

DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number gains and losses and DNA methylation. Advances in high-throughput, genome-wide profiling technologies, such as microarrays, have significantly improved our ability to identify and detect these specific alterations. However as technology continues to improve, a limiting factor remains sample quality and availability. Furthermore, follow-up clinical information and disease outcome are often collected years after the initial specimen collection. Specimens, typically formalin-fixed and paraffin embedded (FFPE), are stored in hospital archives for years to decades. DNA can be efficiently and effectively recovered from paraffin-embedded specimens if the appropriate method of extraction is applied. High quality DNA extracted from properly preserved and stored specimens can support quantitative assays for comparisons of normal and diseased tissues and generation of genetic and epigenetic signatures 1. To extract DNA from paraffin-embedded samples, tissue cores or microdissected tissue are subjected to xylene treatment, which dissolves the paraffin from the tissue, and then rehydrated using a series of ethanol washes. Proteins and harmful enzymes such as nucleases are subsequently digested by proteinase K. The addition of lysis buffer, which contains denaturing agents such as sodium dodecyl sulfate (SDS), facilitates digestion 2. Nucleic acids are purified from the tissue lysate using buffer-saturated phenol and high speed centrifugation which generates a biphasic solution. DNA and RNA remain in the upper aqueous phase, while proteins, lipids and polysaccharides are sequestered in the inter- and organic-phases respectively. Retention of the aqueous phase and repeated phenol extractions generates a clean sample. Following phenol extractions, RNase A is added to eliminate contaminating RNA. Additional phenol extractions following incubation with RNase A are used to remove any remaining enzyme. The addition of sodium acetate and isopropanol precipitates DNA, and high speed centrifugation is used to pellet the DNA and facilitate isopropanol removal. Excess salts carried over from precipitation can interfere with subsequent enzymatic assays, but can be removed from the DNA by washing with 70% ethanol, followed by centrifugation to re-pellet the DNA 3. DNA is re-suspended in distilled water or the buffer of choice, quantified and stored at -20&deg;C. Purified DNA can subsequently be used in downstream applications which include, but are not limited to, PCR, array comparative genomic hybridization 4 (array CGH), methylated DNA Immunoprecipitation (MeDIP) and sequencing, allowing for an integrative analysis of tissue/tumor samples.

This video demonstrates the protocol for DNA extraction from formalin-fixed paraffin-embedded material. This is a multi-day procedure in which tissue sections are deparaffinized with xylene, rehydrated with ethanol and treated with proteinase K to purify and isolate DNA for subsequent gene-specific or genome-wide analysis.

유전과 Epigenetic 분석을위한 파라핀 임베디드 재료에서 DNA 추출

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number ...

Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis

Drosophila has long been used as model system to study development, mainly due to the ease with which it is genetically tractable. Over the years, a plethora of mutant strains and technical tricks have been developed to allow sophisticated questions to be asked and answered in a reasonable amount of time. Fundamental insight into the interplay of components of all known major signaling pathways has been obtained in forward and reverse genetic Drosophila studies. The fly eye has proven to be exceptionally well suited for mutational analysis, since, under laboratory conditions, flies can survive without functional eyes. Furthermore, the surface of the insect eye is composed of some 800 individual unit eyes (facets or ommatidia) that form a regular, smooth surface when looked at under a dissecting microscope. Thus, it is easy to see whether a mutation might affect eye development or growth by externally looking for the loss of the smooth surface ('rough eye' phenotype; Fig. 1) or overall eye size, respectively (for examples of screens based on external eye morphology see e.g.1). Subsequent detailed analyses of eye phenotypes require fixation, plastic embedding and thin-sectioning of adult eyes.
The Drosophila eye develops from the so-called eye imaginal disc, a bag of epithelial cells that proliferate and differentiate during larval and pupal stages (for review see e.g. 2). Each ommatidium consists of 20 cells, including eight photoreceptors (PR or R-cells; Fig. 2), four lens-secreting cone cells, pigment cells ('hexagon' around R-cell cluster) and a bristle. The photoreceptors of each ommatidium, most easily identified by their light sensitive organelles, the rhabdomeres, are organized in a trapezoid made up of the six "outer" (R1-6) and two "inner" photoreceptors (R7/8; R8 [Fig. 2] is underneath R7 and thus only seen in sections from deeper areas of the eye). The trapezoid of each facet is precisely aligned with those of its neighbors and the overall anteroposterior and dorsoventral axes of the eye (Fig. 3A). In particular, the ommatidia of the dorsal and ventral (black and red arrows, respectively) halves of the eye are mirror images of each other and correspond to two chiral forms established during planar cell polarity signaling (for review see e.g. 3).
The method to generate semi-thin eye sections (such as those presented in Fig. 3) described here is slightly modified from the one originally described by Tomlinson and Ready4. It allows the morphological analysis of all cells except for the transparent cone cells. In addition, the pigment of R-cells (blue arrowheads in Fig. 2 and 3) can be used as a cell-autonomous marker for the genotype of a R-cell, thus genetic requirements of genes in a subset of R-cells can readily be determined5,6.

Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis

A standard approach to prepare adult Drosophila eyes for semi-thin sectioning and light microscopic analysis is presented here. The protocol can be used for gross morphological analysis of eye defects, or with the indicated adjustments can be used to determine genetic requirements of genes in specific cell types of the eye (e.g. clonal analysis of photoreceptors) or for electron microscopic analysis.

성인의 준비 Drosophila</em씬 Sectioning 및 현미경 분석을위한> 눈

Drosophila has long been used as model system to study development, mainly due to the ease with which it is genetically tractable. Over the years, a ...

Proteomic Sample Preparation from Formalin Fixed and Paraffin Embedded Tissue

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale quantitative analysis of formalin fixed and paraffin embedded (FFPE) tissue. The procedure comprises four distinct steps. The first one is the preparation of sections from the FFPE material and microdissection of cells of interest. In the second step the isolated cells are lysed and processed using 'filter aided sample preparation' (FASP) technique. In this step, proteins are depleted from reagents used for the sample lysis and are digested in two-steps using endoproteinase LysC and trypsin. 
After each digestion, the peptides are collected in separate fractions and their content is determined using a highly sensitive fluorescence measurement. Finally, the peptides are fractionated on 'pipette-tip' microcolumns. The LysC-peptides are separated into 4 fractions whereas the tryptic peptides are separated into 2 fractions. In this way prepared samples allow analysis of proteomes from minute amounts of material to a depth of 10,000 proteins. Thus, the described workflow is a powerful technique for studying diseases in a system-wide-fashion as well as for identification of potential biomarkers and drug targets.

Archival formalin fixed and paraffin embedded (FFPE) clinical samples are valuable material for investigation of diseases. Here we demonstrate a sample preparation workflow allowing in-depth proteomic analysis of microdissected FFPE tissue.

포르말린에서 프로테오믹스 샘플 준비 파라핀 조직에게 고정 및

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale ...

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

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

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

qPCR에 포르말린 고정, 파라핀 - 임베디드 생검 조직에서 Cytomegaloviral DNA의 검출에 대한 민감하고 빠른 방법입니다

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

Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple method for the immune detection of proteins, the mouse lactating mammary gland being taken as an example. A protocol for the preparation of the tissue samples, especially concerning the dissection of mouse mammary gland, tissue fixation and frozen tissue sectioning, are detailed. A standard protocol to perform indirect immunofluorescence, including an optional antigen retrieval step, is also presented. The observation of the labeled tissue sections as well as image acquisition and post-treatments are also stated. This procedure gives a full overview, from the collection of animal tissue to the cellular localization of a protein. Although this general method can be applied to other tissue samples, it should be adapted to each tissue/primary antibody couple studied.

The indirect immunofluorescence protocol described in this article allows the detection and the localization of proteins in the mouse mammary gland. A complete method is given to prepare mammary gland samples, to perform immunohistochemistry, to image the tissue sections by fluorescence microscopy, and to reconstruct images.

마우스 유선의 냉동 섹션에 간접 면역 형광

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple ...

Employing Digital Droplet PCR to Detect BRAF V600E Mutations in Formalin-fixed Paraffin-embedded Reference Standard Cell Lines

ddPCR is a highly sensitive PCR method that utilizes a water-oil emulsion system. Using a droplet generator, an extracted nucleic acid sample is partitioned into ~20,000 nano-sized, water-in-oil droplets, and PCR amplification occurs in individual droplets. The ddPCR approach is in identifying sequence mutations, copy number alterations, and select structural rearrangements involving targeted genes. Here, we demonstrate the use of ddPCR as a powerful technique for precisely quantitating rare BRAF V600E mutations in FFPE reference standard cell lines, which is helpful in identifying individuals with cancer. In conclusion, ddPCR technique offers the potential to precisely profile the specific rare mutations in different genes in various types of FFPE samples.

The goal of this video is to demonstrate how to perform&#160;automated DNA extraction from formalin-fixed paraffin-embedded (FFPE) reference standard cell lines and digital droplet PCR&#160;(ddPCR) analysis to detect rare mutations in a clinical setting. Detecting mutations in FFPE samples demonstrates the clinical utility of ddPCR in FFPE samples.