We would like to thank Alex Miller for manuscript revisions. This work was supported with the research grants awarded to M.G. from Cure CMD, Struggle Against Muscular Dystrophy (SAM) and Muscular Dystrophy Association (218938).

1. 조직 수확 및 근육 조직의 동결 <ol><li> 이소 플루 란 (2- 클로로 -2- (다이 플루오 로메 톡시) -1,1,1- 트리 플루오로 에탄)의 과다와 마우스를 안락사. 흄 후드에서이 단계를 수행 및 건조기 또는 이소 플루 란에 적신 면봉을 포함하는 벨 항아리로 보조 봉쇄 방법을 사용합니다. </li><li> 단단히 발바닥을 압박하여 죽음을 확인합니다. 이러한 자궁 전위로 안락사의 보조 방법을 사용합니다. </li><li> 근육에 느슨한 머리카락의 부착을 감소시키기 위해 70 % 알코올로 모피 젖은. 같은 # 5로 미세 집게를 사용하여 사지 떨어져 피부 껍질. </li><li> 조심스럽게 아래 뼈 (이 경우에는 전 경골근 (TA)) 관심의 근육을 분리 건에서 시작하여 제거합니다. </li><li> 근육을 절제 10월 (최적 절단 온도 화합물)에 커버 및 라벨 일회용 동결 금형 위에 올려 놓습니다. 근육이 타이에 정상적인 생리적 방향 (머리에 있는지 확인스트레칭없이 L). 대안 적으로, 작은 스티로폼 사각형에 근육을 동결 및 근육의 정확한 길이를 보장하는 곤충 핀을 사용하여 고정. </li><li> 액체 질소를 사용하여 강철 비이커 중 2- 메틸 부탄 (이소 펜탄)을 진정. 5 분 -이 평균 3 일이 걸립니다. 흰색 침전물이 비커의 바닥과 벽에 형성하기 시작할 때까지 간헐적으로 교반하십시오. 주 :이 때, 이소 펜탄 최적 동결 온도에 도달 (-150 ° C) </li><li> 냉장 2- 메틸 부탄에 근육을 포함하는 금형을 찍어. 20 초 - 15 비복근의 가자미근 (GS)과 삼두근을 추천 삼성 전자와 큰 근육 - 같은 다이어프램과 신근 심지 6 longus (EDL)와 같은 작은 근육을 동결. 너무 오래이 균열로 이어질 수 있기 위해 근육을 동결하지 마십시오. </li><li> 드라이 아이스에 냉동 조직을 전송하고 이소 펜탄 증발 (이 과정은 약 평균 15 소요 있습니다 - 20 분)을 할 수 있습니다. 알루미늄 호일에 조직을 감싸고 U에 보관ltra 저온 절편까지 (-70 ° C에서 -80 ° C). </li></ol> 2. 조직 블록을 준비하는 OCT에서 조직의 포함 <ol><li> 드라이 아이스에 전송 조직 해동을 방지하기 위해 cyrostat합니다. 전송 조직하고 30 분 동안 평형화하자 (조직의 완전 해동을 피하기 위해 -20 -24 ℃로 설정)를 챔버 저온 유지한다. </li><li> 펠티에 베이에 냉각십시오. 그라 이오 스탯이 냉각이없는 경우, 신속하게 OCT를 동결 에어로졸 냉각 스프레이를 사용합니다. 이 동결 손상을 초래할 수 있으므로 삽입시에 임의의 지점에서 근육 해동시키지 마십시오. </li><li> 시편 디스크에 10월의 균일 한 박막을 추가하고 식힌다. OCT의 대부분 동안 여전히 액체 상단에 바닥에 고정 될 때, (세로 섹션에 대한 10월에 횡단면과 평행을위한 10월에 수직) 올바른 방향에서 조직을 삽입합니다. 빨리 OCT를 동결 에어로졸 냉각 스프레이를 사용합니다. 열 내선과 조직의 unembeded 부분을 터치ractor 45 초 동안 기다립니다. </li><li> 근육 주위 OCT의 다른 얇은 층을 추가 에어로졸 냉각 스프레이 스프레이 및 이전 단계에서와 같이 열을 추출한다. </li><li> 근육 전체가 커버 될 때까지 분무하고 열 추출기 냉각 에어로졸의 조합을 사용하여 빠르게 동결 OCT를 조금씩 OCT를 계속 추가하고. </li><li> 조직 블록의 상단에 열 추출기를 넣고 5 분 동안 기다린다. </li></ol> 3. 섹션을 준비하고 근육의 미드 벨리 지역에서 섹션을 확인 <ol><li> 시편 머리에 시료를 탑재합니다. 디스크를 보호하기 위해 노브를 조입니다. 시편 디스크 시편 머리와 잘 접촉되어 있는지 확인합니다. </li><li> ° -21 내지 -24 C의 챔버 온도를 조정하고 설정 온도가 될 때까지 기다립니다. </li><li> 두께 설정 (7 μm의)를 조정; 전진 또는 일반 및 미세 설정을 사용하여 블록을 후퇴하여 블록을 잘라. </li><li> 섹션을 수집슬라이드 상에 컷 부의 부착을 용이하게하기 위해 반대 전하 간의 인력을 사용하여, 상기 슬라이드 부에의 상단을 가압하여 양전하 현미경 슬라이드를 미리 예열. </li><li> 섹션의 단면적 (CSA)를 측정하여 중간 배꼽 부분을 확인합니다. 현미경에 연결되어있는 컴퓨터를 사용하여이 촬상 소프트웨어를 수행. 주 : 위상차 이미지를 촬영하고, 전체 둘레의 근육은 소프트웨어 프로그램에 대한 추적 기능을 통해 측정된다. 이것은 CSA 모두뿐만 아니라 최소 족제비 직경 측정 (입자의 양측에 두 개의 평행 한 접선 사이의 최소 거리로 표현 섬유 단면 크기의 측정)을 수득한다. 참고 : CSA 및 최소 흰 족제비 지름 측정은 고원에 시작하면, 중간 배에 도달했습니다. </li><li> 재고는 미래 염색 -80 ° C에서 슬라이드를 보관합니다. 참고 : stainin하는 OCT를 전에 제거 할 필요가 없습니다G 절차. 슬라이드가 coverslipped되면, 그들은 즉시 염색 할 준비가 된 것입니다. </li></ol> 4. 염색 주 : 상세한 단계별 절차가 제품 데이터 시트에서 확인할 수 있고, 염색 절차 따라야하는 동안 개인의 최적화가 최적의 착색을 얻기 위해 필요할 수있다. <ol><li> 미디어 (표 참조)를 장착하는 물과 혼화하지 않기 때문에, (70 %, 95 %, 100 % 에탄올로 2 분마다) 알코올의 증가 성적 통해 슬라이드를 탈수 및 크실렌 (5 분 동안 100 %)을 보장하기 위해 함께 그것들을 취소 슬라이드는 시간이 지남에 따라 불투명가되지 않습니다. </li><li> 섹션 커버 슬립하고 건조 할 수 있습니다. 현미경으로 이미지 이미징 소프트웨어를 장착. </li></ol>

<ol>
	<li>Bonnemann, C. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+approach+to+the+congenital+muscular+dystrophies.">Diagnostic approach to the congenital muscular dystrophies.</a> Neuromuscular disorders : NMD. 24, 289-311 (2014).</li><li>Caughey, J. E., Pachomov, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+diaphragm+in+dystrophia+myotonica.">The diaphragm in dystrophia myotonica.</a> J Neurol Neurosurg Psychiatry. 22, 311-313 (1959).</li><li>Fetterman, G. H., Wratney, M. J., Donaldson, J. S., Danowski, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscular+dystrophy.+I.+History,+clinical+status,+muscle+strength,+and+biopsy+findings.">Muscular dystrophy. I. History, clinical status, muscle strength, and biopsy findings.</a> AMA J Dis Child. 91, 326-338 (1956).</li><li>Platzer, A. C., Chase, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histologic+Alterations+in+Preclinical+Mouse+Muscular+Dystrophy.">Histologic Alterations in Preclinical Mouse Muscular Dystrophy.</a> The American journal of pathology. 44, 931-946 (1964).</li><li>Mercuri, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+magnetic+resonance+imaging+involvement+in+muscular+dystrophies+with+rigidity+of+the+spine.">Muscle magnetic resonance imaging involvement in muscular dystrophies with rigidity of the spine.</a> Ann Neurol. 67, 201-208 (2010).</li><li>Tasca, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+molecular+signatures+in+magnetic+resonance+imaging-staged+facioscapulohumeral+muscular+dystrophy+muscles.">Different molecular signatures in magnetic resonance imaging-staged facioscapulohumeral muscular dystrophy muscles.</a> PloS one. 7, e38779 (2012).</li><li>Girgenrath, M., Dominov, J. A., Kostek, C. A., Miller, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+apoptosis+improves+outcome+in+a+model+of+congenital+muscular+dystrophy.">Inhibition of apoptosis improves outcome in a model of congenital muscular dystrophy.</a> The Journal of clinical investigation. 114, 1635-1639 (2004).</li><li>Kumar, A., Yamauchi, J., Girgenrath, T., Girgenrath, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle-specific+expression+of+insulin-like+growth+factor+1+improves+outcome+in+Lama2Dy-w+mice,+a+model+for+congenital+muscular+dystrophy+type+1A.">Muscle-specific expression of insulin-like growth factor 1 improves outcome in Lama2Dy-w mice, a model for congenital muscular dystrophy type 1A.</a> Human molecular genetics. 20, 2333-2343 (2011).</li><li>Mehuron, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dysregulation+of+matricellular+proteins+is+an+early+signature+of+pathology+in+laminin-deficient+muscular+dystrophy.">Dysregulation of matricellular proteins is an early signature of pathology in laminin-deficient muscular dystrophy.</a> Skeletal muscle. 4, 14 (2014).</li><li>Yamauchi, J., Kumar, A., Duarte, L., Mehuron, T., Girgenrath, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Triggering+regeneration+and+tackling+apoptosis:+a+combinatorial+approach+to+treating+congenital+muscular+dystrophy+type+1+A.">Triggering regeneration and tackling apoptosis: a combinatorial approach to treating congenital muscular dystrophy type 1 A.</a> Human molecular genetics. 22, 4306-4317 (2013).</li><li>Sheriffs, I. N., Rampling, D., Smith, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paraffin+wax+embedded+muscle+is+suitable+for+the+diagnosis+of+muscular+dystrophy.">Paraffin wax embedded muscle is suitable for the diagnosis of muscular dystrophy.</a> Journal of clinical pathology. 54, 517-520 (2001).</li><li>Fox, C. H., Johnson, F. B., Whiting, J., Roller, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formaldehyde+fixation.">Formaldehyde fixation.</a> Journal of Histochemistry &amp; Cytochemistry. 33, 845-853 (1985).</li><li>Maccarty, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Diagnostic+Reliability+of+Frozen+Sections.">The Diagnostic Reliability of Frozen Sections.</a> The American journal of pathology. 5, 377-380 (1929).</li><li>Shi, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+value+of+frozen+tissue+section+used+as+'gold+standard'+for+immunohistochemistry.">Evaluation of the value of frozen tissue section used as 'gold standard' for immunohistochemistry.</a> Am J Clin Pathol. 129, 358-366 (2008).</li><li>Turan, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accuracy+of+frozen+sections+for+intraoperative+diagnosis+of+complex+atypical+endometrial+hyperplasia.">Accuracy of frozen sections for intraoperative diagnosis of complex atypical endometrial hyperplasia.</a> Asian Pacific journal of cancer prevention : APJCP. 13, 1953-1956 (2012).</li></ol>

The authors have nothing to disclose.

조직학 및 면역 조직 화학 다양한 신경 근육 질환의 증상과 병변의 진행을 이해하기위한 주요 도구로 사용되어왔다. 클래식, 근육 생검 먼저 포름 알데히드에 고정 후 파라핀 왁스 내장했다. 포름 알데히드는 고정 조직 구조를 보존하고 RT에서 티슈 저장 및 전송을 허용하지만, 또한 정확한 면역을 달성하기 위해 추가의 단계를 필요로 가교 효소와 단백질을 비활성화한다. 이 문제는 고정 된 섹션?...

근육 조직 학적 검사는 신경근 장애 1-4의 다른 유형의 이해에 중요한 역할을한다. 다른 기술과 결합 할 때, 연구자들은 병리의 증상과 진행의 더 나은 아이디어를 얻을 수 있고 치료 적 개입 5-10의 효능을 평가하는 것이 필수적이 될 수 있습니다. 절단 직전의 생리 상태를 유지하도록하지만 정확한 것으로, 조직을 적절한 방식으로 유지 될 필요가있다. 이것은 제거, 보존, 절편 및 염색시 세심한주의가 필요합니다. 조직은 광학 현미경 연구를위한 두 가지 방법으로 제조 될 수있다. (FFPE) 섹션, 포함 된 첫 번째 방법, 포르말린 고정 파라핀 조직을 파라핀 왁스 (11, 12)에 포함되기 전에 10 % 자연 버퍼 포름 알데히드에 고정해야합니다. 정착 단계는 또한 크로스 링크 내인성 PR 나은 형태를 유지하는 데 도움oteins 따라서 면역 항원에 대한 복잡한 검색 절차를 필요로하며 12 절에 효소 염색 가능성을 제거. 냉동 된 부분은, 다른 한편으로는, 미리 고정 된 또는 처리 될 필요가 없다; 따라서, 단백질은 고유 생물 입체 (13, 14)을 보유 할 수 있습니다. 또한, 동결 절편은 또한 때때로 육종 및 기타 외과 생검 (15)의 진단 수술, 예를 들어 필요한 빠른 처리 시간을 허용. 제대로 수행하지 않을 경우 그러나,이 방법은 조직 학적 검사에 대한 부분은 부적당하고 근육 구조에 대한 변경 사항을 소개 손상을 동결하는 경향이있다. 이 논문은 적절한 검사를위한 최적의 염색을 달성하기 위해 근육 조직 검사를 준비하는 가장 효과적인 방법을 개설 할 것이다.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Cryostat</td>
			<td>Leica CM 1850&#160;</td>
			<td>Leica Microsystems Inc.</td>
			<td>Buffalo Grove, IL 60089 United States, office phone 1-800 248 0123 
			Richard Allen Scientific</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Nikon Eclipse 50i&#160;</td>
			<td>Nikon Instruments Inc.</td>
			<td>1300 Walt Whitman Road, Melville, N.Y. 11747-3064, U.S.A. 
			Phone: +1-631-547-8500 
			Olympus</td>
		</tr>
		<tr>
			<td>Image analysis software</td>
			<td>Nikon Imaging Software Basic Research</td>
			<td>Nikon Instruments Inc.</td>
			<td>1300 Walt Whitman Road, Melville, N.Y. 11747-3064, U.S.A. 
			Phone: +1-631-547-8500 
			Image J, Metamorph</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>14043-220-05</td>
			<td>JD Medical Dist. Co., Inc.</td>
			<td>JD Medical Distrubuting Co., Inc 1923 West Peoria Avenue Phoenix, Arizona 85029</td>
		</tr>
		<tr>
			<td>OCT</td>
			<td>4583</td>
			<td>Sakura Inc.</td>
			<td>Sakrua Fintek, USA, Torrence, CA 90501, USA 
			Leica Microsystem</td>
		</tr>
		<tr>
			<td>Freeze It</td>
			<td>23-022524</td>
			<td>Thermofisher</td>
			<td>790 Memorial Dr, Cambridge, MA, USA, 02139 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>Disposable tissue mold</td>
			<td>22-363-554</td>
			<td>Thermofisher</td>
			<td>790 Memorial Dr, Cambridge, MA, USA, 02139 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>Colorfrost plus slides</td>
			<td>9991001</td>
			<td>Thermofisher</td>
			<td>790 Memorial Dr, Cambridge, MA, USA, 02139 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>650501-4L</td>
			<td>Sigma-Aldrich</td>
			<td>3050 Spruce Street, St.Louis MO, 63103 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>Ethyl alcohol</td>
			<td>HC-1300-1GL</td>
			<td>Thermofisher</td>
			<td>790 Memorial Dr, Cambridge, MA, USA, 02139 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>2-methyl butane</td>
			<td>M 32631-SL</td>
			<td>Sigma-Aldrich</td>
			<td>3050 Spruce Street, St.Louis MO, 63103 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>HC-7001GA</td>
			<td>Thermofisher</td>
			<td>790 Memorial Dr, Cambridge, MA, USA, 02139 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>Cytoseal 280</td>
			<td>8311-4</td>
			<td>Thermofisher</td>
			<td>790 Memorial Dr, Cambridge, MA 02139 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>Hematoxylin Gill #3</td>
			<td>CS402-1D</td>
			<td>Thermofisher</td>
			<td>790 Memorial Dr, Cambridge, MA, USA, 02139 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>Eosin</td>
			<td>6766007</td>
			<td>Thermofisher</td>
			<td>790 Memorial Dr, Cambridge, MA, USA, 02139 
			VWR Scientific</td>
		</tr>
	</tbody>
</table>

do's and don'ts in the preparation of muscle cryosections for histological analysis

근육 생검의 조직 학적 평가는 신경 근육 질환의 병리의 개발 및 진행의​​ 이해에 필수적인 도구 역임했다. 그러나, 이렇게하기 위해, 적절한 치료는 절제 최적 염색을 달성하기 위해 조직을 보존 할 때 수행되어야한다. 조직 보존의 한 방법은 파라핀 왁스를 삽입 한 후 포름 알데히드의 조직을 고정하고 포함한다. 이 방법은 잘 형태를 유지하고 실온에서 장기간 보관이 가능하지만, 복잡하고 독성 화학 물질의 취급을 요구한다. 또한, 효과적인 면역 항원에 대한 검색 프로토콜을 필요 항원 가교, 포름 알데히드 고정 결과. 반대로, 동결 절편은 고정을 필요로하며, 따라서 생물 항원 형태를 유지하지 않는다. 이 방법은 intraopera 같은 빠른 조직 학적 평가를 필요로하는 상황에서이 특히 유용하고, 시간의 주위에 빠른 차례에 뚜렷한 장점을 제공합니다수술 적 생검을 포지티브,. 여기에서 우리는 다른 조직 학적 및 면역 학적 얼룩과 시각화를위한 근육 조직 검사를 준비하는 가장 효과적인 방법을 설명합니다.

수확 및 조직의 동결을위한 셋업은 그림 1에서 볼 수 있습니다. 절제된 조직은 건조 (그림 1A)를 방지하기 위해 PBS에 적신 거즈에 저장해야합니다. 조직들은 정확한 형태 (도 1b)을 보장하기 위해 OCT에서 침지 크라이 금형에 뻗어 올바른 방향으로 가능한 생리적 길이에 가까워 야 학적 해석에 사용하기 위해 선택되는 경우. 동결 조직 적합 펜탄 슬러리는 액체 ?...

Watch this Scientific Journal Video about Do's and Don'ts in the Preparation of Muscle Cryosections for Histological Analysis at JoVE.com

Do's and Don'ts in the Preparation of Muscle Cryosections for Histological Analysis

Here we demonstrate the most efficient methods for freezing, embedding, cryosectioning, and staining of muscle biopsies to avoid freezing artifacts.

조직 학적 분석을위한 근육 저온부의 준비의 것과하지 말아야을 수행

Histological evaluation of muscle biopsies has served as an indispensable tool in the understanding of the development and progression of pathology of ...

do-s-don-ts-preparation-muscle-cryosections-for-histological

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31.7K 조회수.  Boston University. Here we demonstrate the most efficient methods for freezing, embedding, cryosectioning, and staining of muscle biopsies to avoid freezing artifacts.

비디오: Do's and Don'ts in the Preparation of Muscle Cryosections for Histological Analysis

Myo-mechanical Analysis of Isolated Skeletal Muscle

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force generation and fatigability in treated muscle. We describe a detailed approach to evaluating myo-mechanical properties in freshly explanted hindlimb muscle from the mouse. We describe the atraumatic harvest of mouse extensor digitorum longus muscle, mounting the muscle in a muscle strip myograph (Model 820MS; Danish Myo Technology), and the measurement of maximal twitch and tetanic tension, contraction time, and half-relaxation time, using a square pulse stimulator (Model S48; Grass Technologies). Using these measurements, we demonstrate the calculation of specific twitch and tetanic tension normalized to muscle cross-sectional area, the twitch-to-tetanic tension ratio, the force-frequency relationship curve and the low frequency fatigue curve 4. This analysis provides a method for quantitative comparison between therapeutic interventions in mouse models of muscle disease 1,2,3,5, as well as comparison of the effects of genetic modification on muscle function 6,7,8,9.

To assess the in vivo effects of therapeutic interventions for muscle disease, methods are needed to quantitate force generation and fatigability in treated muscle. We detail an approach to evaluating myo-mechanical properties in explanted mouse hindlimb muscle. This analysis provides a robust approach to quantitating the effects of genetic modification on muscle function, as well as comparison of therapies in mouse models of muscle disease.

절연 골격근의 묘 - 기계 분석

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force ...

연구

의학

In Vivo Canine Muscle Function Assay

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric 
contractions. The pelvic limb of an anesthetized dog is immobilized in a stereotactic frame to align the tibia at a right angle to the femur. Adhesive wrap affixes the 
paw to a pedal mounted on the shaft of a servomotor to measure torque. Percutaneous nerve stimulation activates pelvic limb muscles of the paw to either push (extend) or 
pull (flex) against the pedal to generate isometric torque. Percutaneous tibial nerve stimulation activates tibiotarsal extensor muscles. Repeated eccentric (lengthening) 
contractions are induced in the tibiotarsal flexor muscles by percutaneous peroneal nerve stimulation. The eccentric protocol consists of an initial isometric contraction 
followed by a forced stretch imposed by the servomotor. The rotation effectively lengthens the muscle while it contracts, e.g., an eccentric contraction. During 
stimulation flexor muscles are subjected to an 800 msec isometric and 200 msec eccentric contraction. This procedure is repeated every 5 sec. To avoid fatigue, 4 min rest 
follows every 10 contractions with a total of 30 contractions performed.

In Vivo Canine Muscle Function Assay

We describe a minimally-invasive and painless method to measure canine hindlimb muscle strength and muscle response to repeated eccentric contractions.

VIVO 개 근육 기능 분석에

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric 
...

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases in children and adults. These antibodies have been used to guide diagnosis and treatment. Cell-based assays have improved the detection of antibodies in patient serum. They are based on the surface expression of brain antigens on eukaryotic cells, which are then incubated with diluted patient sera followed by fluorochrome-conjugated secondary antibodies. After washing, secondary antibody binding is then analyzed by flow cytometry. Our group has developed a high-throughput flow cytometry live cell-based assay to reliably detect antibodies against specific neurotransmitter receptors. This flow cytometry method is straight forward, quantitative, efficient, and the use of a high-throughput sampler system allows for large patient cohorts to be easily assayed in a short space of time. Additionally, this cell-based assay can be easily adapted to detect antibodies to many different antigenic targets, both from the central nervous system and periphery. Discovering additional novel antibody biomarkers will enable prompt and accurate diagnosis and improve treatment of immune-mediated disorders.

Over the recent years, live cell-based assays have been used successfully to detect antibodies against surface and conformational antigens. Here, we describe a method using high-throughput flow cytometry enabling the analysis of large cohorts of patients. Detection of novel antibodies will improve diagnosis and treatment of immune-mediated disorders.

인간 혈청에있는 N-메틸-D-아스파 라진 산염 수용체 또는 도파민 2 수용체에 대한 항체를 검출하는 높은 처리량 유동 세포 계측법 세포 기반 분석

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

Collection and Extraction of Saliva DNA for Next Generation Sequencing

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality DNA. However, DNA extraction from saliva can yield high quality DNA with little to no degradation/fragmentation that is suitable for a variety of DNA assays without the expense of a phlebotomist and can even be acquired through the mail. However, at present, no saliva DNA collection/extraction protocols for next generation sequencing have been presented in the literature. This protocol optimizes parameters of saliva collection/storage and DNA extraction to be of sufficient quality and quantity for DNA assays with the highest standards, including microarray genotyping and next generation sequencing.

DNA extraction from saliva can provide a readily available source of high molecular weight DNA, with little to no degradation/fragmentation. This protocol provides optimized parameters for saliva collection/storage and DNA extraction to be of sufficient quality and quantity for downstream DNA assays with high quality requirements.

차세대 시퀀싱에 대한 수집 및 타액 DNA의 추출

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Ultrasound Based Assessment of Coronary Artery Flow and Coronary Flow Reserve Using the Pressure Overload Model in Mice

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow reserve (CFR) in humans. Reduced CFR is accompanied by marked intramyocardial and pericoronary fibrosis and is used as an indication of the severity of dysfunction. This study explores, step-by-step, the real-time changes measured in the coronary flow velocity, CFR and systolic to diastolic peak velocity (S/D) ratio in the setting of an aortic banding model in mice. By using a Doppler transthoracic imaging technique that yields reproducible and reliable data, the method assesses changes in flow in the septal coronary artery (SCA), for a period of over two weeks in mice, that previously either underwent aortic banding or thoracotomy. 
During imaging, hyperemia in all mice was induced by isoflurane, an anesthetic that increased coronary flow velocity when compared with resting flow. All images were acquired by a single imager. Two ratios, (1) CFR, the ratio between hyperemic and baseline flow velocities, and (2) systolic (S) to diastolic (D) flow were determined, using a proprietary software and by two independent observers. Importantly, the observed changes in coronary flow preceded LV dysfunction as evidenced by normal LV mass and fractional shortening (FS). 
The method was benchmarked against the current gold standard of coronary assessment, histopathology. The latter technique showed clear pathologic changes in the coronary artery in the form of peri-coronary fibrosis that correlated to the flow changes as assessed by echocardiography. 
The study underscores the value of using a non-invasive technique to monitor coronary circulation in mouse hearts. The method minimizes redundant use of research animals and demonstrates that advanced ultrasound-based indices, such as CFR and S/D ratios, can serve as viable diagnostic tools in a variety of investigational protocols including drug studies and the study of genetically modified strains.

Coronary flow reserve (CFR) is useful for assessment of myocardial oxygen demand and evaluation of cardiovascular risk. This study establishes a step-by-step transthoracic Doppler echocardiographic (TTDE) method for longitudinal monitoring of the changes in CFR, as measured from coronary artery in mice, under the experimental pressure overload of aortic banding.

마우스의 압력 과부하 모델을 사용하여 관상 동맥 흐름 및 관상 동맥 흐름 준비 제도 이사회의 초음파를 바탕으로 평가

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow ...

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and well tolerated technique. The Weil-Blakesley conchotome has a sharp biting tip with a 4 - 6 mm wide hollow. It is inserted through a 5 - 10 mm skin incision and can be maneuvered for controlled tissue penetration. The tip is opened and closed within the tissue and then rotated through 90 -180&#176; to cut the muscle. The amount of muscle obtained following repeated sampling can vary from 20 mg to 290 mg which can be processed for both histology and molecular studies. The wound needs to be kept dry and vigorous physical activity kept to a minimum for approximately 72 hr although normal levels of activity can restart immediately following the procedure. This procedure is safe and effective when close attention is paid to the selection of subjects, full asepsis and post procedure care. &#160;Both right and left vastus lateralis are suitable for biopsy dependent on participant preference.

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

This video demonstrates the technique of percutaneous muscle biopsy of the human vastus lateralis using the Weil-Blakesley conchotome.

사람의<em&gt; 광근의 Lateralis</em&gt; 웨일 - Blakesley Conchotome를 사용하여 골격 근육 생검

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and ...

Calcification of Vascular Smooth Muscle Cells and Imaging of Aortic Calcification and Inflammation

Cardiovascular disease is the leading cause of morbidity and mortality in the world. Atherosclerotic plaques, consisting of lipid-laden macrophages and calcification, develop in the coronary arteries, aortic valve, aorta, and peripheral conduit arteries and are the hallmark of cardiovascular disease. In humans, imaging with computed tomography allows for the quantification of vascular calcification; the presence of vascular calcification is a strong predictor of future cardiovascular events. Development of novel therapies in cardiovascular disease relies critically on improving our understanding of the underlying molecular mechanisms of atherosclerosis. Advancing our knowledge of atherosclerotic mechanisms relies on murine and cell-based models. Here, a method for imaging aortic calcification and macrophage infiltration using two spectrally distinct near-infrared fluorescent imaging probes is detailed. Near-infrared fluorescent imaging allows for the ex vivo quantification of calcification and macrophage accumulation in the entire aorta and can be used to further our understanding of the mechanistic relationship between inflammation and calcification in atherosclerosis. Additionally, a method for isolating and culturing animal aortic vascular smooth muscle cells and a protocol for inducing calcification in cultured smooth muscle cells from either murine aortas or from human coronary arteries is described. This in vitro method of modeling vascular calcification can be used to identify and characterize the signaling pathways likely important for the development of vascular disease, in the hopes of discovering novel targets for therapy.

Vascular calcification is an important predictor of and contributor to human cardiovascular disease. This protocol describes methods for inducing calcification of cultured primary vascular smooth muscle cells and for quantifying calcification and macrophage burden in animal aortas using near-infrared fluorescence imaging.

대동맥 석회화와 염증의 혈관 평활근 세포 및 이미지의 석회화

Cardiovascular disease is the leading cause of morbidity and mortality in the world.  Atherosclerotic plaques, consisting of lipid-laden macrophages and ...

Dissection of the Mouse Pancreas for Histological Analysis and Metabolic Profiling

We have been investigating the pancreas specific transcription factor, 1a cre-recombinase; lox-stop-lox- Kristen rat sarcoma, glycine to aspartic acid at the 12 codon (Ptf1acre/+;LSL-KrasG12D/+) mouse strain as a model of human pancreatic cancer. The goal of our current studies is to identify novel metabolic biomarkers of pancreatic cancer progression. We have performed metabolic profiling of urine, feces, blood, and pancreas tissue extracts, as well as histological analyses of the pancreas to stage the cancer progression. The mouse pancreas is not a well-defined solid organ like in humans, but rather is a diffusely distributed soft tissue that is not easily identified by individuals unfamiliar with mouse internal anatomy or by individuals that have little or no experience performing mouse organ dissections. The purpose of this article is to provide a detailed step-wise visual demonstration to guide novices in the removal of the mouse pancreas by dissection. This article should be especially valuable to students and investigators new to research that requires harvesting of the mouse pancreas by dissection for metabolic profiling or histological analyses.

This video article provides a detailed demonstration of the procedures required to successfully remove the pancreas from a mouse by dissection for histological analysis and metabolic profiling.

조직학 분석 및 대사 프로 파일링에 대 한 마우스 췌의 해 부

We have been investigating the pancreas specific transcription factor, 1a cre-recombinase; lox-stop-lox- Kristen rat sarcoma, glycine to aspartic acid at ...

Echocardiographic and Histological Examination of Cardiac Morphology in the Mouse

An increasing number of genetically modified mouse models has become available in recent years. Moreover, the number of pharmacological studies performed in mice is high. Phenotypic characterization of these mouse models also requires the examination of cardiac function and morphology. Echocardiography and magnetic resonance imaging (MRI) are commonly used approaches to characterize cardiac function and morphology in mice. Echocardiographic and MRI equipment specialized for use in small rodents is&#160;expensive and requires a dedicated space. This protocol describes cardiac measurements in mice using a clinical echocardiographic system with a 15 MHz human vascular probe. Measurements are performed on anesthetized adult mice. At least three image sequences are recorded and analyzed for each animal in M-mode in the parasternal short-axis view. Afterwards, cardiac histological examination is performed, and cardiomyocyte diameters are determined on hematoxylin-eosin- or wheat germ agglutinin (WGA)-stained paraffin sections. Vessel density is determined morphometrically after Pecam-1 immunostaining. The protocol has been applied successfully to pharmacological studies and different genetic animal models under baseline conditions, as well as after experimental myocardial infarction by the permanent ligation of the left anterior descending coronary artery (LAD). In our experience, echocardiographic investigation is limited to anesthetized animals and is feasible in adult mice weighing at least 25 g.

Echocardiographic examination is frequently used in mice. Expensive high-resolution ultrasound devices have been developed for this purpose. This protocol describes an affordable echocardiographic procedure combined with histological morphometric analyses to determine cardiac morphology.

마우스의 심장 형태학의 echocardiographic 및 조직학 검사

An increasing number of genetically modified mouse models has become available in recent years. Moreover, the number of pharmacological studies performed ...