본 연구는 크리스찬 도플러 연구 재단에 의해 지원되었다. 우리는 프로젝트 전반에 걸쳐 지원 비엔나, 오스트리아의 의과 대학의 핵심 시설 이미지에서 사빈 Rauscher에게 감사의 말씀을 전합니다. 차 항체는 NIH의 NICHD에 의해 만들어지고 아이오와, 생물학과, 아이오와 시티, 아이오와 대학에서 유지, 발달 연구 하이 브리 도마 은행에서 얻은, Schiaffino, S.에 의해 개발되었다.

FELASA (26)에 의해 권장하는 동물 과목을 포함한 모든 절차는 실험 동물 관리의 원칙을 준수하여 실시 하였다. 승인 비엔나의 의과 대학과 연구 및 과학에 대한 오스트리아 정부의 제도적 검토위원회에 의해 연구 이전에 얻은 (BMWF는 : BMWF - 66.009 / 0222-WF / II / 3B / : Bundesministerium은 Wissenschaft 놀이 Forschung, 참조 번호 FUER 2014). 1. 근육 수확 참고 : 멩 등의 알 의해 이전 간행물. 도 27에 자세히 근육 시료의 정확한 동결 설명 가능하다. <ol><li> 즉시 동물의 안락사 후 전체 단면을 확보하기 위해 전체 근육 또는 충분한 조직을 얻습니다. <ol><li> 마취 또는 안락사 쥐에서 전체 근육을 제거합니다. 모든 결합 조직과 집게와 가위로 근육을 둘러싸고있는 힘줄을 제거합니다. A에 대한nesthesia 또는 안락사는 FELASA (28)의 국제 지침을 따르십시오. </li></ol></li><li> 교정 정밀 저울을 사용하여 근육의 무게를. 이 빠른 단계는 특히 중재 절차 후 근육 샘플 간의 비교 분석을 할 수 있습니다. </li><li> 모든면에 약 1mm의 안전 마진 근육의 크기에 따라,의 OCT 화합물로 완전히 충전 된 용기에 넣고 근육 (예를 들어, 알루미늄 박으로 이루어지는). </li><li> 약 2 분에 동결 액체 질소를 이용하여 이소 펜탄을 미리 냉각. 이소 펜탄은 용기의 하단에 백색 결정을 보여주기 시작해야합니다. 참고 :이 단계는 근육의 정확한 구조를 유지에 필수적인하여 올바른 염색을 허용하고 섬유 (27)를 분석합니다. </li><li> 스토어 샘플은 -80 ° C에서 최소 24 시간 동안 OCT에서 보존. 샘플은, 그러나, corre 경우, 몇 달 또는 몇 년 -80 ° C에 저장 될 수있다<html> ctly 냉각. </li><li> cryotome를 사용하여 -20 ℃에서 근육의 중앙부에서 10 개 μm의 횡단면을 잘라. 을 10㎛ 절단 가능한 것은 아닌 경우, 20 ㎛의 최대까지, 2㎛의 단계 두께를 증가시킨다. 충분히 예리한 절삭 날이 단계는 필수적이다. </li><li> 단면에 슬라이드를 근사에 의해 접착제 슬라이드에 섹션을 적용합니다. 크로스 섹션은 자동 접착 슬라이드에 충실 할 것이다. 하룻밤 염색하기 전에 -20 ° C에서 슬라이드를 저장합니다. </li></ol> 2. 염색 참고 : 마이 오신 중쇄 단백질에 대한 항체의 많은 수를 사용할 수 있습니다; 그러나, 높은 품질의 항체는 자동 수집 및 분석을 위해 필수적이다. 항체 희석을 참고로, 표 1 참조. 스프레드 시트 파일이 올바른 희석을 계산하고 필요한 솔루션 수량의 수를 확인하려면, 참조1 파일 source.jove.com/files/ftp_upload/55441/Calculator_for_muscle_fiber_typing.xlsx&quot;&gt;Supplemental. <ol><li> 해동 염색 과정 전에 10 분 동안 냉동 된 근육 부분을 공기 건조. </li><li> 워시 인산염 완충 식염수 (PBS) + 0.05 % 트리톤 X 10 분 후, 5 분 동안 조심스럽게 슬라이드. 트리톤은 크게 염색 품질을 향상시키기 위해 표시되었습니다. 자세한 내용은 설명을 참조하십시오. </li><li> 2 분 구간 공기 건조하자 (항체의 필요한 양을 최소화하기 위해), 15 분 동안 추가 건조를 허용 소수성 펜을 사용하여 각 슬라이드의 개별 단면 개요. </li><li> PBS + 모든 슬라이드 차단 0.05 % 트리톤 X (PBST)을 실온에서 1 시간 동안 10 %의 염소 혈청을 함유. </li><li> PBS + 0.05 % 트리톤 X (PBST)을 함유하는 10 %의 염소 혈청 칵테일 모든 일차 항체 (표 1) 희석. 신청하기 전에 충분히 섞는다. 트리톤 X는 전체 단면의 동일한 염색을 위해 필수적이다. 계산하다단면 당 일차 항체 칵테일의 30 μL. </li><li> 단계 2.7-2.13를 들어 광으로부터 충분한 보호를 보장합니다. 흐리게 실내 조명 커버와 염색 트레이 적절한 보호를 제공한다. 또, 염색시에 습윤 환경을 제공하고, 건조를 방지하기 위해 하단에서 유체에 의한 염색 트레이를 채운다. </li><li> 실온에서 습기 염색 트레이에서 1 시간 동안 슬라이드에 차 항체 (표 1) 칵테일을 적용한다. </li><li> 5 분 동안 새로운 PBST로 다음 10 분 동안 PBS + 0.05 % 트리톤 X와 슬라이드를 씻어. </li><li> PBS + 0.05 % 트리톤 X (PBST) + 10 % 염소 혈청의 칵테일 이차 항체를 희석. 단면 당 일차 항체 칵테일 중 30 μL를 계산한다. </li><li> 1 시간 동안 슬라이드에 차 항체 칵테일을 적용합니다. </li><li> PBS로 씻어 슬라이드 + 0.05 % 트리톤 X 및 10 분 동안 추가 5 분 동안 새로운 PBST로 (PBST). </li><li> DAPI 핵 염색 키트 (바람직하게 읽기에 사용 솔루션) FO 적용R 15 분 또는 세포 핵을 얼룩 제조업체의 지침에 따라. </li><li> 워시 간단히 PBS + 0.05 %와 슬라이드 트리톤 X 1 분 (PBST). 그 후, 슬라이드를 자연 건조 간단하게 할 수 있습니다. </li><li> 형광등 설치 매체와 커버 슬립을 적용합니다. 4 ℃에서 보관 빛으로부터 보호하고 24-48 시간 내에 최적의 촬상을 수행한다. </li></ol> 3. 현미경 <ol><li> 로드 슬라이드 스캐너에 슬라이드. 슬라이드 (12)의 최대 슬라이드 스캐너에 따라 가능하다. </li><li> 새 프로젝트를 시작하십시오. 미리보기를 들어, DAPI 채널과 2.5 배 렌즈를 설정합니다. 자세한 분석을 위해 DAPI, GFP, FITC와 Cy5의 채널 및 20X 대물 렌즈를 사용한다. 자동 초점은 DAPI 채널에서 프로젝트를 통해 획득된다. </li><li> DAPI의 파란, FITC : 녹색, 텍사스 레드 : 빨강, Cy5의 : 노란색 각 채널에 대해 다음 색상을 사용합니다. </li><li> DAPI 채널을 사용하여 각 슬라이드의 미리보기를 만듭니다. 이를 위해 첫 번째 시료에 수동 초점을 적용하고 미리보기를 통해 사용분석 결과 모든 시료의 미리보기 이미지를 수집합니다. </li><li> 자세한 획득 과정에 포함되어야한다 각 단면을 설명합니다. 관심의 전체 영역의 캡션을 보장하기 위해, 시편의 가장자리에 설명 그릴하지 마십시오. </li><li> 각 채널에 대한 노출 시간을 설정합니다. 대부분의 경우, 서로 다른 채널의 노광 파라미터를 모든 단면에 대해 동일하다. </li><li> 자동 이미지 수집을 실행합니다. 초기 자동화 과정에서 잘못된 인수를 방지하기 위해, 뷰의 첫 번째 필드에 대한 올바른 인수를 확인합니다. </li><li> 필요한 경우, 데스크톱 소프트웨어 미러링을 사용하여 원격 제어를 설정합니다. 이것은 어떤 네트워크 또는 인터넷 연결을 통해 다른 컴퓨터로 슬라이드 스캐너의 컴퓨터 화면을 미러링하여 영상 획득 과정의 원격 모니터링을 허용한다. 변경이 사용 된 슬라이드에 대한 물리적 설정 이루어지지 할 수 있지만, 이미지 수집 소프트웨어의 가상 환경은 모니터링 할 수 있습니다<html> 이미지의 정확한 초점이나 노출 시간. 또한, 완료 예상 시간을 정기적으로 확인할 수 있습니다. </li><li> 화상 획득의 종료 후, 섬유 구조, 개개의 섬유 및 섬유 다양한 유형 구별하는 경우 수동으로 제어하여 모든 이미지의 정확한 자동 초점 제어한다. </li><li> 다시 취득 잘못보기 또는 관심의 전체 영역의 단일 필드에 대한 이미지 영역을 집중했다. 단일 영역의 재 획득을 위해, 전체 프로젝트 전반에 걸쳐 영역이나 이미지를 표시 및 수동 초점 영역을 재 취득. 대부분의 경우, 다른 초점 수준의 추가 이미지 세부는 잘못 초점을 맞춘 이미지로 연결 </li><li> 최종 분석에 포함되어야 할 때마다 단면의 경우, JPEG 파일로 개별적으로 (DAPI 제외한) 각 채널을 내보내. 이름을 지정하고 텍사스 레드, FITC와 Cy5에라는 이름의 폴더에 노출 된 채널에 따라 파일을 정렬 할 수 있습니다. </li></ol> 4. 자동 섬유 분석 십t &quot;&gt; 참고 : 매크로는 다음 웹 페이지에서 얻을 수 있습니다 : https://www.meduniwien.ac.at/hp/bionicreconstruction/macro/ <ol><li> 미리 분석 전에 종래의 화상 편집 소프트웨어를 사용하여 염색하고 섬유와 배경 간의 충분한 대조 확인 각 이미지. 필요한 경우, 밝기 및 콘트라스트 조정 명령을 사용하여, 그 차이를 증가 대비 및 밝기를 조정한다. </li><li> 열기 ImageJ에 또는 피지. 명령을 사용하여 매크로를 열고 &quot;- 매크로 - 플러그인. 편집&quot; 이 매크로 소스 코드를 보여줍니다 및 매개 변수의 빠른 적응을 할 수 있습니다. 필요한 경우, 매크로의 소스 코드에서 각 채널에 대한 폴더 디렉토리를 변경합니다. 참고 : 근육 섬유 분석에 대한 값은 쥐 팔뚝과 lumbrical 근육, 다른 종 또는 다른 값을 요구할 수있다 근육을 기반으로합니다. </li><li> 매크로를 시작합니다 &quot;실행&quot;명령을 사용하십시오. 폴더의 모든 이미지는 이제 매크로에로드하고 연속적인 순서로 정량화된다. 결과는 우입니다새 창에서 WN. 이 단계는 데이터가 분석되는 양에 따라 몇 시간이 초까지 걸릴 수있다. </li><li> 스프레드 시트 파일로 결과 창을 내 보냅니다. (느리게) 긍정적으로 계산 Cy5에 대한 (중간) FITC와 텍사스 레드 (빠른) 섬유를 값을 확인하고 전체 섬유 번호 요약. </li></ol>

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Res. 383, 140-151 (2001).</li><li>Schiaffino, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibre+types+in+skeletal+muscle:+a+personal+account.">Fibre types in skeletal muscle: a personal account.</a> Acta Physiol (Oxf). 199 (4), 451-463 (2010).</li><li>Bottinelli, R., Betto, R., Schiaffino, S., Reggiani, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unloaded+shortening+velocity+and+myosin+heavy+chain+and+alkali+light+chain+isoform+composition+in+rat+skeletal+muscle+fibres.">Unloaded shortening velocity and myosin heavy chain and alkali light chain isoform composition in rat skeletal muscle fibres.</a> J Physiol. 478, 341-349 (1994).</li><li>Schiaffino, S., Reggiani, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myosin+isoforms+in+mammalian+skeletal+muscle.">Myosin isoforms in mammalian skeletal muscle.</a> J Appl Physiol (1985). 77 (2), 493-501 (1994).</li><li>Larsson, L., Moss, R. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+methods+for+the+analysis+of+skeletal+muscle+fiber+size+and+metabolic+type.">Automated methods for the analysis of skeletal muscle fiber size and metabolic type.</a> Int Rev Cell Mol Biol. 306, 275-332 (2013).</li><li>Schiaffino, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three+myosin+heavy+chain+isoforms+in+type+2+skeletal+muscle+fibres.">Three myosin heavy chain isoforms in type 2 skeletal muscle fibres.</a> J Muscle Res Cell Motil. 10 (3), 197-205 (1989).</li><li>Lieber, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Skeletal muscle structure, function, and plasticity. , (2009).</li><li>Hintz, C. S., Coyle, E. F., Kaiser, K. K., Chi, M. M., Lowry, O. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+muscle+fiber+typing+by+quantitative+enzyme+assays+and+by+myosin+ATPase+staining.">Comparison of muscle fiber typing by quantitative enzyme assays and by myosin ATPase staining.</a> J Histochem Cytochem. 32 (6), 655-660 (1984).</li><li>Havenith, M. G., Visser, R., van Schendel, J. M. S. c. h. r. i. j. v. e. r. s. -., Bosman, F. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+fiber+typing+in+routinely+processed+skeletal+muscle+with+monoclonal+antibodies.">Muscle fiber typing in routinely processed skeletal muscle with monoclonal antibodies.</a> Histochemistry. 93 (5), 497-499 (1990).</li><li>Likar, B., Pernu&#353;, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Registration+of+serial+transverse+sections+of+muscle+fibers.">Registration of serial transverse sections of muscle fibers.</a> Cytometry. 37 (2), 93-106 (1999).</li><li>Liu, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+fiber-type-specific+cross-sectional+area+assessment+and+myonuclei+counting+in+skeletal+muscle.">Automated fiber-type-specific cross-sectional area assessment and myonuclei counting in skeletal muscle.</a> J Appl Physiol (1985). 115 (11), 1714-1724 (2013).</li><li>Bloemberg, D., Quadrilatero, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+determination+of+myosin+heavy+chain+expression+in+rat,+mouse,+and+human+skeletal+muscle+using+multicolor+immunofluorescence+analysis.">Rapid determination of myosin heavy chain expression in rat, mouse, and human skeletal muscle using multicolor immunofluorescence analysis.</a> PLoS One. 7 (4), 35273 (2012).</li><li>Bergmeister, K. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+muscle+fiber+type+population+analysis+with+ImageJ+of+whole+rat+muscles+using+rapid+myosin+heavy+chain+immunohistochemistry.">Automated muscle fiber type population analysis with ImageJ of whole rat muscles using rapid myosin heavy chain immunohistochemistry.</a> Muscle Nerve. 54 (2), 292-299 (2016).</li><li>Guillen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FELASA+guidelines+and+recommendations.">FELASA guidelines and recommendations.</a> J Am Assoc Lab Anim Sci. 51 (3), 311-321 (2012).</li><li>Meng, H., 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=Tissue+Triage+and+Freezing+for+Models+of+Skeletal+Muscle+Disease.">Tissue Triage and Freezing for Models of Skeletal Muscle Disease.</a> J Vis Exp. (89), e51586 (2014).</li><li>Guillen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FELASA+Guidelines+and+Recommendations.">FELASA Guidelines and Recommendations.</a> J Am Assoc Lab Animal Sci. 51 (3), 311-321 (2012).</li><li>Ribari&#269;, S., &#268;eba&#352;ek, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+Visualization+of+Myosin+Heavy+Chain+Isoforms+in+Single+Muscle+Sections.">Simultaneous Visualization of Myosin Heavy Chain Isoforms in Single Muscle Sections.</a> Cells Tissues Organs. 197 (4), 312-321 (2013).</li></ol>

The authors have nothing to disclose.

<html> 여기, 우리가 공부하고 자동으로 시간을 효율적으로 면역 조직 화학 염색을 통해 쥐의 단면의 근육 섬유의 인구를 정량화하기 위해 광범위하게 접근 방법을 보여줍니다. 재현성을 위해, 우리는 단계의 설명과 본 연구에서 설명되지 않은 다른 종의 응용 프로그램에 대한 잠재적 인 수정하여 자세한 단계를 제시한다. 또한, 우리는 최적의 기능과 한계에 대한 절차의 장점, 전제 조건에 대해 설?...

골격근 조성물은 노화 1, 2, 운동 3, 4, 5, 6, 7, 또는 질병 8, 9, 10 또는 11 같은 외상 병태 생리적 과정 과정에서 큰 변화를 겪는다. 따라서, 이러한 프로세스의 구조적 효과에 대한 연구 집중의 여러 분야는 기능 변화를 이해합니다. 근육의 기능을 결정하는 핵심 요소 중 하나는 근섬유의 조성물이다. 근섬유 다른 마이 오신 중쇄 (MHC) 단백질을 발현시켜, 13, 중간 느린 또는 빠른 섬유 (7), (12)로 구분 </sup<html> &gt; 14, 15, 16, 17. 생리 학적, 근육은 몸에 그 기능에 따라 다른 근육 섬유의 조성이있다. 근섬유 입력을 사용하여, 섬유 집단은 17 생리 학적 또는 병태 생리 학적 과정 7 적응력을 식별하기 위해 정량화 될 수있다. 역사적으로, 시간이 많이 소요되는 다수의 방법은 근육 섬유 유형을 구별하기 위해 적용되어왔다. 이를 위해, 근육 섬유는 다양한 pH 값 또는 근육 효소 활성에 미오신의 ATPase의 반응성에 의해 어느 분류 하였다. 다른 섬유의 특성은 하나의 섹션에서 평가 될 수없는 것처럼, 여러 단면이 모든 근육 섬유를 확인하고 정량화 14, 16, 17 수동 있도록해야했습니다,= &quot;외부 참조&quot;&gt; 18, 19, 20, 21, 22. 대조적으로, 최근 간행물 빠르게 한 단면에서 여러 종류의 섬유를 염색 미오신 중쇄 단백질에 대한 면역 조직 화학 (IHC)를 사용 하였다. 이 절차의 장점을 바탕으로, 지금은 근육 섬유 인구 분석 19, 23, 24의 황금 표준으로 간주됩니다. 개선 IHC 염색 프로토콜을 사용하여, 우리는 전체 근육의 단면 이후 자동 근육 섬유 정량의 완전 자동 인수는 오픈 소스 플랫폼 ImageJ에를 사용 가능한 것으로 보여 최근 수 있었다. 수동 정량에 비해, 우리의 방법은 4 % ~ ± 정확하면서 슬라이드 당 필요한 시간 (수동의 약 10 %를 분석)의 상당한 감소를 제공하는 25 </s&gt;까지. 이 방법의 전반적인 목표는 오픈 소스 플랫폼을 사용하여 전체 쥐의 근육에서 자동 근육 섬유 정량에 신속하고 신뢰할 수있는, 사용자가 독립적 인 가이드를 설명하는 것입니다. 또한, 우리는 쥐 또는 인간의 근육과 같은 다른 시편의 사용을 허용 할 가능성이 수정 사항을 설명합니다.

<table border="0" cellpadding="0" cellspacing="0" width="986">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">O.C.T compound</td>
			<td style="width:397px;">Tissue-Tek, Sakura, Netherlands</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">For embedding of muscle tissue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isopentane</td>
			<td></td>
			<td></td>
			<td>for adequate freezing of muscle tissue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Superfrost Ultra Plus slides</td>
			<td>Thermo Scientific, Germany</td>
			<td align="right">1014356190</td>
			<td>adhesive slides</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">phosphate buffered saline&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100</td>
			<td>Thermo Scientific, Germany</td>
			<td align="right">85112</td>
			<td>Detergent Soluation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat serum</td>
			<td>Thermo Scientific, Germany</td>
			<td>50197Z</td>
			<td>Goat Serum</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DAKO Fluorescent Mounting Medium</td>
			<td>Dako Denmark</td>
			<td>S3023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dako pen</td>
			<td>Dako Denmark</td>
			<td>S200230-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TissueFAXSi plus&#160;</td>
			<td>TissueGnostics, Vienna, Austria</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Primary antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MHC-I (Cat# BA-F8, RRID: AB_10572253)</td>
			<td>Developmental Studies Hybridoma Bank (DSHB, Iowa, USA)</td>
			<td></td>
			<td>Supernatant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MHC-IIa (Cat# SC-71, RRID: AB_2147165)</td>
			<td>Developmental Studies Hybridoma Bank (DSHB, Iowa, USA)</td>
			<td></td>
			<td>Supernatant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MHC-IIb (Cat# BF-F3, RRID: AB_2266724)</td>
			<td>Developmental Studies Hybridoma Bank (DSHB, Iowa, USA)</td>
			<td></td>
			<td>Supernatant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Secondary antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa&#160;Fluor&#160;633&#160;Goat Anti-Mouse IgG2b&#160;</td>
			<td>Thermo Scientific, Germany</td>
			<td>A-21146</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 488 Goat Anti-Mouse IgG1&#160;(&#947;1)</td>
			<td>Thermo Scientific, Germany</td>
			<td>A-21121</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 555 Goat Anti-Mouse IgM (&#181; chain),</td>
			<td>Thermo Scientific, Germany</td>
			<td>A-21426</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NucBlue Fixed Cell ReadyProbes Reagent</td>
			<td>Thermo Scientific, Germany</td>
			<td>R37606</td>
			<td></td>
		</tr>
	</tbody>
</table>

a rapid automated protocol for muscle fiber population analysis in rat muscle cross sections using myosin heavy chain immunohistochemistry

근육 섬유 인구의 정량화 질환, 외상 및 골격 근육의 구성에 다양한 영향의 효과에 대한 깊은 통찰력을 제공합니다. 다양한 시간이 많이 걸리는 방법은 전통적으로 연구의 많은 분야에서 섬유 인구를 연구하는 데 사용되었다. 그러나, 최근에 하나의 섹션에서 여러 섬유 종류를 식별하기 위해 빠른 대안을 제공 마이 오신 중쇄 단백질 발현에 따라 면역 조직 화학적 방법을 개발했다. 여기, 우리는 전체 크로스 섹션과 ImageJ에와 섬유 인구의 자동 정량을 자동으로 얻을 수 있도록 개선 염색 품질에 대한 신속하고 안정적이고 재현 가능한 프로토콜을 제시한다. 이를 위해, 임베디드 골격 근육 세포 핵 염색을위한 이차 형광 항체 및 DAPI와 미오신 중쇄 항체를 사용하여 염색 단면으로 절단된다. 전체 단면은 고해상도의 복합체를 획득하기 위하여 슬라이드 스캐너를 사용하여 자동으로 스캔전체 표본의 사진. 섬유 인구 분석은 이후 ImageJ에 대한 자동화 된 매크로를 사용, 느린 중간 빠른 섬유를 정량화하기 위해 수행된다. 우리는 이전에이 방법은 ± 4 % 정도에 안정적으로 섬유 인구를 식별 할 수있는 것으로 나타났습니다. 또한,이 방법은 사용자 간 변동과 크게 오픈 소스 ImageJ에 플랫폼을 사용하여 분석 당 시간을 감소시킨다.

전체 쥐의 근육 단면은 MHC I, IIA 및 IIB 근육 섬유를 식별하기 위해 면역 조직 화학 염색을 사용하여 빠른 속도로 염색 하였다. 형광 현미경 슬라이드 스캐너를 사용하여, 전체 단면이 자동 자동 근섬유 취득 하였다은 ImageJ에로 분석한다. 절차의 개념은 근육 섬유의 정량화를위한 ​​간단하고 신뢰성 및 시간 효율적인 워크 플로우를 제공하는 기반으로합니다. <p class="jo...

Watch this Scientific Journal Video about A Rapid Automated Protocol for Muscle Fiber Population Analysis in Rat Muscle Cross Sections Using Myosin Heavy Chain Immunohistochemistry at JoVE.com

A Rapid Automated Protocol for Muscle Fiber Population Analysis in Rat Muscle Cross Sections Using Myosin Heavy Chain Immunohistochemistry

여기에서는 개선 된 염색 품질함으로써 자동 취득하고 자유롭게 사용할 수있는 소프트웨어를 사용 ImageJ에 섬유 집단의 정량화를 허용 빠른 근섬유 분석을위한 프로토콜을 제시한다.

마이 오신 중쇄 면역를 사용하여 쥐의 근육 크로스 섹션에서 근육 섬유 인구 분석을위한 신속한 자동화 프로토콜

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle ...

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Biology

11.2K 조회수.  Medical University of Vienna.  여기에서는 개선 된 염색 품질함으로써 자동 취득하고 자유롭게 사용할 수있는 소프트웨어를 사용 ImageJ에 섬유 집단의 정량화를 허용 빠른 근섬유 분석을위한 프로토콜을 제시한다. 

비디오: A Rapid Automated Protocol for Muscle Fiber Population Analysis in Rat Muscle Cross Sections Using Myosin Heavy Chain Immunohistochemistry

Immunohistochemistry on Paraffin Sections of Mouse Epidermis Using Fluorescent Antibodies

In the epidermis, immunohistochemistry is an efficient means of localizing specific proteins to their relative expression compartment; namely the basal, suprabasal, and stratum corneum layers. The precise localization within the epidermis of a particular protein lends clues toward its functional role within the epidermis. In this chapter, we describe a reliable method for immunolocalization within the epidermis modified for both frozen and paraffin sections that we use very routinely in our laboratory. Paraffin sections generally provide much better morphology, hence, superior results and photographs; however, not all antibodies will work with the harsh fixation and treatment involved in their processing. Therefore, the protocol for frozen sectioning is also included. Within paraffin sectioning, two fixation protocols are described (Bouin's and paraformaldehyde); the choice of fixative will be directly related to the antibody specifications and may require another fixing method.

We describe a reliable method for immunolocalization within the epidermis modified for both frozen and paraffin sections that we use very routinely in our laboratory.

형광 항체를 사용하여 마우스 표피의 파라핀 섹션에 Immunohistochemistry

In the epidermis, immunohistochemistry is an efficient means of localizing specific proteins to their relative expression compartment; namely the basal, ...

연구

생물학

Procedures for Rat in situ Skeletal Muscle Contractile Properties

There are many circumstances where it is desirable to obtain the contractile response of skeletal muscle under physiological circumstances: normal circulation, intact whole muscle, at body temperature. This includes the study of contractile responses like posttetanic potentiation, staircase and fatigue. Furthermore, the consequences of disease, disuse, injury, training and drug treatment can be of interest. This video demonstrates appropriate procedures to set up and use this valuable muscle preparation.
To set up this preparation, the animal must be anesthetized, and the medial gastrocnemius muscle is surgically isolated, with the origin intact. Care must be taken to maintain the blood and nerve supplies. A long section of the sciatic nerve is cleared of connective tissue, and severed proximally. All branches of the distal stump that do not innervate the medial gastrocnemius muscle are severed. The distal nerve stump is inserted into a cuff lined with stainless steel stimulating wires. The calcaneus is severed, leaving a small piece of bone still attached to the Achilles tendon. Sonometric crystals and/or electrodes for electromyography can be inserted. Immobilization by metal probes in the femur and tibia prevents movement of the muscle origin. The Achilles tendon is attached to the force transducer and the loosened skin is pulled up at the sides to form a container that is filled with warmed paraffin oil. The oil distributes heat evenly and minimizes evaporative heat loss. A heat lamp is directed on the muscle, and the muscle and rat are allowed to warm up to 37&deg;C. While it is warming, maximal voltage and optimal length can be determined. These are important initial conditions for any experiment on intact whole muscle. The experiment may include determination of standard contractile properties, like the force-frequency relationship, force-length relationship, and force-velocity relationship.
With care in surgical isolation, immobilization of the origin of the muscle and alignment of the muscle-tendon unit with the force transducer, and proper data analysis, high quality measurements can be obtained with this muscle preparation.

Procedures for Rat in situ Skeletal Muscle Contractile Properties

This video demonstrates the surgical preparation and procedures needed to study the contractile responses of the rat medial gastrocnemius muscle preparation in situ. This preparation allows measurement of skeletal muscle contractile properties under physiological conditions. The animal is anesthetized and the muscle is separated from surrounding tissue at its distal end. The Achilles tendon is attached to a force transducer, allowing measurement of the muscle&#x2019;s contractile response at 37 degrees C with an intact circulation.

쥐 절차<em&gt; 현장에서</em&gt; 골격근 수축 등록

There are many circumstances where it is desirable to obtain the contractile response of skeletal muscle under physiological circumstances: normal ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

라이브 세포의 저장소로 작동하는 칼슘 항목의 형광 기반의 측정 : 교양 암 세포에서 골격근 섬유에

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Analysis of Skeletal Muscle Defects in Larval Zebrafish by Birefringence and Touch-evoke Escape Response Assays

Zebrafish (Danio rerio) have become a particularly effective tool for modeling human diseases affecting skeletal muscle, including muscular dystrophies1-3, congenital myopathies4,5, and disruptions in sarcomeric assembly6,7, due to high genomic and structural conservation with mammals8. Muscular disorganization and locomotive impairment can be quickly assessed in the zebrafish over the first few days post-fertilization. Two assays to help characterize skeletal muscle defects in zebrafish are birefringence (structural) and touch-evoked escape response (behavioral).
Birefringence is a physical property in which light is rotated as it passes through ordered matter, such as the pseudo-crystalline array of muscle sarcomeres9. It is a simple, noninvasive approach to assess muscle integrity in translucent zebrafish larvae early in development. Wild-type zebrafish with highly organized skeletal muscle appear very bright amidst a dark background when visualized between two polarized light filters, whereas muscle mutants have birefringence patterns specific to the primary muscular disorder they model. Zebrafish modeling muscular dystrophies, diseases characterized by myofiber degeneration followed by repeated rounds of regeneration, exhibit degenerative dark patches in skeletal muscle under polarized light. Nondystrophic myopathies are not associated with necrosis or regenerative changes, but result in disorganized myofibers and skeletal muscle weakness. Myopathic zebrafish typically show an overall reduction in birefringence, reflecting the disorganization of sarcomeres.
The touch-evoked escape assay involves observing an embryo&#39;s swimming behavior in response to tactile stimulation10-12. In comparison to wild-type larvae, mutant larvae frequently display a weak escape contraction, followed by slow swimming or other type of impaired motion that fails to propel the larvae more than a short distance12. The advantage of these assays is that disease progression in the same fish type can be monitored in vivo for several days, and that large numbers of fish can be analyzed in a short time relative to higher vertebrates.

The zebrafish is now an established and powerful tool for modeling muscular dystrophies, congenital myopathies, and related neuromuscular diseases. Birefringence and touch-evoked escape behavior are two common noninvasive assays used to determine the degree of muscular disorganization and locomotive impairment of zebrafish embryos during early development.

Zebrafish (Danio rerio) have become a particularly effective tool for modeling human diseases affecting skeletal muscle, including muscular ...

Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ring-supported hydrophilized PTFE membrane to provide structural support to both living and fixed tissue during immunohistochemical processing, which allows for the use of a variety of protocols that would otherwise cause damage to the tissue. First, this is demonstrated with bolus loading of fluorescent markers into living retinal tissue. This method allows for quick visualization of targeted structures, while the membrane support maintains tissue integrity during the injection and allows for easy transfer of the preparation for further imaging or processing.
Second, a procedure for antibody staining in tissue fixed with carbodiimide is described. Though paraformaldehyde fixation is more common, carbodiimide fixation provides a superior substrate for the visualization of synaptic proteins. A limitation of carbodiimide is that the resulting fixed tissue is relatively fragile; however, this is overcome with the use of the supporting membrane. Retinal tissue is used to demonstrate these techniques, but they may be applied to any fragile tissue.

The protocol described here is for structural assessment of a wholemount retinal preparation. This includes descriptions of tissue dissection, mounting onto a hydrophilized polytetrafluoroethylene (PTFE) membrane insert, bolus loading with fluorescent markers, and a comparison of fixation with carbodiimide and paraformaldehyde for immunohistochemical analysis of cellular and synaptic components.

이미징 및 면역 조직 화학에 대한 망막 Wholemount 준비를 위해 최적화 된 프로토콜

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ...

Generation of Myospheres From hESCs by Epigenetic Reprogramming

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based therapies and for a "disease in a dish" model of human neuromuscular diseases. Major hurdles, such as low abundance and heterogeneity of the population of interest, as well as a lack of protocols for the formation of three-dimensional contractile structures, have limited the applications of stem cells for neuromuscular disorders. We have designed a protocol that overcomes these limits by ectopic introduction of defined factors in hESCs - the muscle determination factor MyoD and SWI/SNF chromatin remodeling complex component BAF60C - that are able to reprogram hESCs into skeletal muscle cells. Here we describe the protocol established to generate hESC-derived myoblasts and promote their clustering into tridimensional miniaturized structures (myospheres) that functionally mimic miniaturized skeletal muscles7.

Here, we describe a protocol based on epigenetic reprogramming of human embryonic stem cells (hESCs) toward generating a homogeneous population of skeletal muscle progenitors that under permissive culture conditions form three-dimensional clusters of contractile myofibers (myospheres), which recapitulate biological features of human skeletal muscles.

성적인 프로그램을 다시 만들에 의해 인간 배아 줄기 세포에서 Myospheres의 생성

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based ...

Visualization and Quantification of the Cell-free Layer in Arterioles of the Rat Cremaster Muscle

The cell-free layer is defined as the parietal plasma layer in the microvessel flow, which is devoid of red blood cells. The measurement of the in vivo cell-free layer width and its spatiotemporal variations can provide a comprehensive understanding of hemodynamics in microcirculation. In this study, we used an intravital microscopic system coupled with a high-speed video camera to quantify the cell-free layer widths in arterioles in vivo. The cremaster muscle of Sprague-Dawley rats was surgically exteriorized to visualize the blood flow. A custom-built imaging script was also developed to automate the image processing and analysis of the cell-free layer width. This approach enables the quantification of spatiotemporal variations more consistently than previous manual measurements. The accuracy of the measurement, however, partly depends on the use of a blue filter and the selection of an appropriate thresholding algorithm. Specifically, we evaluated the contrast and quality of images acquired with and without the use of a blue filter. In addition, we compared five different image histogram-based thresholding algorithms (Otsu, minimum, intermode, iterative selection, and fuzzy entropic thresholding) and illustrated the differences in their determination of the cell-free layer width.

This study demonstrates the surgical preparation of the rat cremaster muscle for the visualization of the in vivo cell-free layer. Considerable factors affecting the accuracy of the cell-free layer width measurement are discussed in this study.

쥐 Cremaster 근육의 세동맥의 셀이없는 계층의 시각화 및 정량화

The cell-free layer is defined as the parietal plasma layer in the microvessel flow, which is devoid of red blood cells. The measurement of the in vivo ...

Optical Cross-Sectional Muscle Area Determination of Drosophila Melanogaster Adult Indirect Flight Muscles

Muscle mass wasting, known as muscle atrophy, is a common phenotype in Drosophila models of neuromuscular diseases. We have used the indirect flight muscles (IFMs) of flies, specifically the dorso-longitudinal muscles (DLM), as the experimental subject to measure the atrophic phenotype brought about by different genetic causes. In this protocol, we describe how to embed fly thorax muscles for semi thin sectioning, how to obtain a good contrast between muscle and the surrounding tissue, and how to process optical microscope images for semiautomatic acquisition of quantifiable data and analysis. We describe three specific applications of the methodological pipeline. First, we show how the method can be applied to quantify muscle degeneration in a myotonic dystrophy fly model; second, measurement of muscle cross-sectional area can help to identify genes that either promote or prevent muscle atrophy and/or muscle degeneration; third, this protocol can be applied to determine whether a candidate compound is able to significantly modify a given atrophic phenotype induced by a disease-causing mutation or by an environmental trigger.

Optical Cross-Sectional Muscle Area Determination of Drosophila Melanogaster Adult Indirect Flight Muscles

We report a method to quantify muscle area, which is an indirect method to determine muscle mass in Drosophila adults. We demonstrate the application of our methodology by analyzing the indirect flight muscles in a Drosophila model of Myotonic Dystrophy disease.

초파리 Melanogaster 성인 간접 비행 근육의 광 단면 근육 면적 결정

Muscle mass wasting, known as muscle atrophy, is a common phenotype in Drosophila models of neuromuscular diseases. We have used the indirect flight ...

Sequential Immunofluorescence and Immunohistochemistry on Cryosectioned Zebrafish Embryos

Investigation of intercellular interactions often requires discrete labeling of specific cell populations and precise protein localization. The zebrafish embryo is an excellent tool for examining such interactions with an in vivo model. Whole-mount immunohistochemical and immunofluorescence assays are frequently applied in zebrafish embryos to assess protein expression. However, it can be difficult to achieve accurate mapping of co-localized proteins in three-dimensional space. In addition, some studies may require the use of two antibodies that are not compatible with the same technique (e.g., antibody 1 is only suitable for immunohistochemistry and antibody 2 is only suitable for immunofluorescence). The purpose of the method described herein is to perform sequential immunofluorescence and/or immunohistochemistry on individual cryosections derived from early-stage zebrafish embryos. Here we describe the use of sequential rounds of immunofluorescence, imaging, immunohistochemistry, imaging for a single cryosection in order to achieve precise identification of protein expression at the single-cell level. This methodology is suitable for any study in early-stage zebrafish embryos that requires accurate identification of multiple protein targets in individual cells.

This protocol demonstrates sequential immunofluorescence and immunohistochemistry on cryosections from early-stage zebrafish embryos enabling precise colocalization analyses in specific cell populations.

냉동 제브라피시 배아의 순차적 면역형광 및 면역성화학

Investigation of intercellular interactions often requires discrete labeling of specific cell populations and precise protein localization. The zebrafish ...

Isolation of Human Ventricular Cardiomyocytes from Vibratome-Cut Myocardial Slices

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly isolated by coronary perfusion with digestive enzymes. However, isolating human cardiomyocytes is challenging because human myocardial specimens usually do not allow for coronary perfusion, and alternative isolation protocols result in poor yields of viable cells. In addition, human myocardial specimens are rare and only regularly available at institutions with on-site cardiac surgery. This hampers the translation of findings from animal to human cardiomyocytes. Described here is a reliable protocol that enables efficient isolation of ventricular myocytes from human and animal myocardium. To increase the surface-to-volume ratio while minimizing cell damage, myocardial tissue slices 300 &#181;m thick are generated from myocardial specimens with a vibratome. Tissue slices are then digested with protease and collagenase. Rat myocardium was used to establish the protocol and quantify yields of viable, calcium-tolerant myocytes by flow-cytometric cell counting. Comparison with the commonly used tissue-chunk method showed significantly higher yields of rod-shaped cardiomyocytes (41.5 &#177; 11.9 vs. 7.89 &#177; 3.6%, p &#60; 0.05). The protocol was translated to failing and non-failing human myocardium, where yields were similar as in rat myocardium and, again, markedly higher than with the tissue-chunk method (45.0 &#177; 15.0 vs. 6.87 &#177; 5.23 cells/mg, p &#60; 0.05). Notably, with the protocol presented it is possible to isolate reasonable numbers of viable human cardiomyocytes (9&#8211;200 cells/mg) from minimal amounts of tissue (&#60;50 mg). Thus, the method is applicable to healthy and failing myocardium from both human and animal hearts. Furthermore, it is possible to isolate excitable and contractile myocytes from human tissue specimens stored for up to 36 h in cold cardioplegic solution, rendering the method particularly useful for laboratories at institutions without on-site cardiac surgery.

Presented is a protocol for the isolation of human and animal ventricular cardiomyocytes from vibratome-cut myocardial slices. High yields of calcium-tolerant cells (up to 200 cells/mg) can be obtained from small amounts of tissue (&#60;50 mg). The protocol is applicable to myocardium exposed to cold ischemia for up to 36 h.

진동절단 심근슬라이스에서 인간 심실 심근세포의 분리

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly ...