Name: do's and don'ts in the preparation of muscle cryosections for histological analysis
Uploaded: 2015-05-15T13:00:00.000+00:00
Duration: 10 min 27 s
Description: Watch this Scientific Journal Video about Do's and Don'ts in the Preparation of Muscle Cryosections for Histological Analysis at JoVE.com

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>

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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><tbody><tr><td>Cryostat</td><td>Leica CM 1850</td><td>Leica Microsystems Inc.</td><td>Buffalo Grove, IL 60089 U.S.A., office phone 1-800-248-0123&lt;br /&gt;Alternative: Richard Allen Scientific</td></tr><tr><td>Microscope</td><td>Nikon Eclipse 50i</td><td>Nikon Instruments Inc.</td><td>1300 Walt Whitman Road, Melville, N.Y. 11747-3064 U.S.A., Phone: 1-631-547-8500&lt;br /&gt;Alternative: 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&lt;br /&gt;Alternatives: Image J, Metamorph</td></tr><tr><td>Isoflurane</td><td>14043-220-05</td><td>JD Medical Dist. Co., Inc.</td><td>1923 West Peoria Avenue, Phoenix, AZ 85029 U.S.A.</td></tr><tr><td>OCT</td><td>4583</td><td>Sakura Inc.</td><td>Torrence, CA 90501 U.S.A.&lt;br /&gt;Alternative source: Leica Microsystem</td></tr><tr><td>Freeze It</td><td>23-022524</td><td>Thermofisher</td><td>790 Memorial Dr, Cambridge, MA, 02139 U.S.A.&lt;br /&gt;Alternative source: VWR Scientific</td></tr><tr><td>Disposable tissue mold</td><td>22-363-554</td><td>Thermofisher</td><td>790 Memorial Dr, Cambridge, MA 02139 U.S.A.&lt;br /&gt;Alternative source: VWR Scientific</td></tr><tr><td>Colorfrost plus slides</td><td>9991001</td><td>Thermofisher</td><td>790 Memorial Dr, Cambridge, MA 02139 U.S.A.&lt;br /&gt;Alternative source: VWR Scientific</td></tr><tr><td>Acetone</td><td>650501-4L</td><td>Sigma-Aldrich</td><td>3050 Spruce Street, St. Louis, MO 63103 U.S.A.&lt;br /&gt;Alternative source: VWR Scientific</td></tr><tr><td>Ethyl alcohol</td><td>HC-1300-1GL</td><td>Thermofisher</td><td>790 Memorial Dr, Cambridge, MA 02139 U.S.A.&lt;br /&gt;Alternative source: 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 U.S.A.&lt;br /&gt;Alternative source: VWR Scientific</td></tr><tr><td>Xylene</td><td>HC-7001GA</td><td>Thermofisher</td><td>790 Memorial Dr, Cambridge, MA 02139 U.S.A.&lt;br /&gt;Alternative source: VWR Scientific</td></tr><tr><td>Cytoseal 280</td><td>8311-4</td><td>Thermofisher</td><td>790 Memorial Dr, Cambridge, MA 02139 U.S.A.&lt;br /&gt;Alternative source: VWR Scientific</td></tr><tr><td>Hematoxylin Gill #3</td><td>CS402-1D</td><td>Thermofisher</td><td>790 Memorial Dr, Cambridge, MA 02139 U.S.A.&lt;br /&gt;Alternative source: VWR Scientific</td></tr><tr><td>Eosin</td><td>6766007</td><td>Thermofisher</td><td>790 Memorial Dr, Cambridge, MA 02139 U.S.A.&lt;br /&gt;Alternative source: 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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31.8K 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

Induction of Acute Skeletal Muscle Regeneration by Cardiotoxin Injection

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced skeletal muscle regeneration is a widely used, powerful model system to study the events involved in muscle regeneration as well as the mechanisms and different players. Indeed, a detailed knowledge of this process is essential for a better understanding of the pathological conditions that lead to skeletal muscle degeneration, and it aids in identifying new targeted therapeutic strategies. The present work describes a detailed and reproducible protocol to induce acute skeletal muscle regeneration in mice through a single intramuscular injection of cardiotoxin (CTX). CTX belongs to the family of snake venom toxins and causes myolysis of myofibers, which eventually triggers the regeneration events. The dynamics of skeletal muscle regeneration is evaluated by histological analysis of muscle sections. The protocol also illustrates the experimental procedures for dissecting, freezing, and cutting the Tibialis Anterior muscle, as well as the routine Hematoxylin &#38; Eosin staining that is widely used for subsequent morphological and morphometric analysis.

This manuscript describes a detailed protocol to induce acute skeletal muscle regeneration in adult mice and subsequent manipulations of the muscles, such as dissection, freezing, cutting, routine staining, and myofiber cross-sectional area analysis.

Cardiotoxin 주입에 의한 급성 골격 근육 재생 유도

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced ...

연구

발생학

Preparation of the Rat Vocal Fold for Neuromuscular Analyses

The purpose of this tutorial is to describe the preparation of the rat vocal fold for histochemical neuromuscular study. This protocol outlines procedures for rat laryngeal dissection, flash-freezing, and cryosectioning of the vocal folds. This study describes how to cryosection vocal folds in both longitudinal and cross-sectional planes. A novelty of this protocol is the laryngeal tracking during cryosectioning that ensures accurate identification of the intrinsic laryngeal muscles and reduces the chance of tissue loss. Figures demonstrate the progressive cryosectioning in both planes. Twenty-nine rat hemi-larynges were cryosectioned and tracked from the emergence of the thyroid cartilage to the appearance of the first section that included the full vocal fold. The full vocal fold was visualized for all animals in both planes. There was high variability in the distance from the appearance of the thyroid cartilage to the appearance of the full vocal fold in both planes. Weight was not correlated to depth of laryngeal landmarks, suggesting individual variability and other factors related to tissue preparation may be responsible for the high variability in the appearance of landmarks during sectioning. This study details a methodology and presents morphological data for preparing the rat vocal fold for histochemical neuromuscular investigation. Due to high individual variability, laryngeal landmarks should be closely tracked during cryosectioning to prevent oversectioning tissue and tissue loss. The use of a consistent methodology, including adequate tissue preparation and awareness of landmarks within the rat larynx, will assist with consistent results across studies and aid new researchers interested in using the rat vocal fold as a model to investigate laryngeal neuromuscular mechanisms.

This protocol describes methods used to prepare rat vocal folds for histochemical neuromuscular study.

신경 근육 분석을위한 쥐 보컬 폴드의 준비

The purpose of this tutorial is to describe the preparation of the rat vocal fold for histochemical neuromuscular study. This protocol outlines procedures ...

신경과학

Tissue Triage and Freezing for Models of Skeletal Muscle Disease

Skeletal muscle is a unique tissue because of its structure and function, which requires specific protocols for tissue collection to obtain optimal results from functional, cellular, molecular, and pathological evaluations. Due to the subtlety of some pathological abnormalities seen in congenital muscle disorders and the potential for fixation to interfere with the recognition of these features, pathological evaluation of frozen muscle is preferable to fixed muscle when evaluating skeletal muscle for congenital muscle disease. Additionally, the potential to produce severe freezing artifacts in muscle requires specific precautions when freezing skeletal muscle for histological examination that are not commonly used when freezing other tissues. This manuscript describes a protocol for rapid freezing of skeletal muscle using isopentane (2-methylbutane) cooled with liquid nitrogen to preserve optimal skeletal muscle morphology. This procedure is also effective for freezing tissue intended for genetic or protein expression studies. Furthermore, we have integrated our freezing protocol into a broader procedure that also describes preferred methods for the short term triage of tissue for (1) single fiber functional studies and (2) myoblast cell culture, with a focus on the minimum effort necessary to collect tissue and transport it to specialized research or reference labs to complete these studies. Overall, this manuscript provides an outline of how fresh tissue can be effectively distributed for a variety of phenotypic studies and thereby provides standard operating procedures (SOPs) for pathological studies related to congenital muscle disease.

The analysis of skeletal muscle tissues to determine structural, functional, and biochemical properties is greatly facilitated by appropriate preparation. This protocol describes appropriate methods to prepare skeletal muscle tissue for a broad range of phenotyping studies.

조직의 우선 순위 구분과 골격근 질환 모델에 대한 동결

Skeletal muscle is a unique tissue because of its structure and function, which requires specific protocols for tissue collection to obtain optimal ...

생물학

Cryosectioning Method for Microdissection of Murine Colonic Mucosa

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The colonic epithelium is dynamically turned over and epithelial cells are generated in the stem cell containing crypts of Lieberk&#252;hn. Progenitor cells produced in the crypt-bases migrate toward the luminal surface, undergoing a process of cellular differentiation before being shed into the gut lumen. In order to study these processes at the molecular level, we have developed a simple method for the microdissection of two spatially distinct regions of the colonic mucosa; the proliferative crypt zone, and the differentiated surface epithelial cells. Our objective is to isolate specific crypt and surface epithelial cell populations from mouse colonic mucosa for the isolation of RNA and protein.

Here we describe a simple method for the isolation of spatially distinct murine colonic epithelial surface cells from the underlying crypt-base cells.

쥐 대장 점막의 이외에 Microdissection을위한 냉동 절편 방법

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The ...

Fiber Type and Subcellular-Specific Analysis of Lipid Droplet Content in Skeletal Muscle

Skeletal muscle lipid infiltration, known as myosteatosis, increases with obesity and ageing. Myosteatosis has also recently been discovered as a negative prognostic factor for several other disorders such as cardiovascular disease and cancer. Excessive lipid infiltration decreases muscle mass and strength. It also results in lipotoxicity and insulin resistance depending on total intramyocellular lipid content, lipid droplet (LD) morphology, and subcellular distribution. Fiber type (oxidative vs glycolytic) is also important, since oxidative fibers have a greater capacity to utilize lipids. Because of their crucial implications in pathophysiology, in-depth studies on LD dynamics and function in a fiber type-specific manner are warranted.
Herein, a complete protocol is presented for the quantification of intramyocellular lipid content and analysis of LD morphology and subcellular distribution in a fiber type-specific manner. To this end, serial muscle cryosections were stained with the fluorescent dye Bodipy and antibodies against myosin heavy chain isoforms. This protocol enables the simultaneous processing of different muscles, saving time and avoiding possible artifacts and, thanks to a personalized macro created in Fiji, the automatization of LD analysis is also possible.

Increasing evidence indicates that excessive infiltration of lipids inside skeletal muscle results in lipotoxicity and diabetes. Here, we present a complete protocol, including tissue processing, staining with Bodipy, image acquisition, and analysis, to quantify the size, density, and subcellular distribution of lipid droplets in a fiber-type specific manner.

골격근의 지질 액적 함량의 섬유 유형 및 세포내 특이적 분석

Skeletal muscle lipid infiltration, known as myosteatosis, increases with obesity and ageing. Myosteatosis has also recently been discovered as a negative ...

의학

High-Throughput, Multi-Image Cryohistology of Mineralized Tissues

There is an increasing need for efficient phenotyping and histopathology of a variety of tissues. This phenotyping need is evident with the ambitious projects to disrupt every gene in the mouse genome. The research community needs rapid and inexpensive means to phenotype tissues via histology. Histological analyses of skeletal tissues are often time consuming and semi-quantitative at best, regularly requiring subjective interpretation of slides from trained individuals. Here, we present a cryohistological paradigm for efficient and inexpensive phenotyping of mineralized tissues. First, we present a novel method of tape-stabilized cryosectioning that preserves the morphology of mineralized tissues. These sections are then adhered rigidly to glass slides and imaged repeatedly over several rounds of staining. The resultant images are then aligned either manually or via computer software to yield composite stacks of several layered images. The protocol allows for co-localization of numerous molecular signals to specific cells within a given section. In addition, these fluorescent signals can be quantified objectively via computer software. This protocol overcomes many of the shortcomings associated with histology of mineralized tissues and can serve as a platform for high-throughput, high-content phenotyping of musculoskeletal tissues moving forward.

In this manuscript, we present a high-throughput, semi-automated cryohistology platform to produce aligned composite images of multiple response measures from several rounds of fluorescent imaging on frozen sections of mineralized tissues.

높은 처리량, 광물 조직의 멀티 이미지 Cryohistology

There is an increasing need for efficient phenotyping and histopathology of a variety of tissues. This phenotyping need is evident with the ambitious ...

Cryosectioning of Contiguous Regions of a Single Mouse Skeletal Muscle for Gene Expression and Histological Analyses

With this method, consecutive cryosections are collected to enable both microscopy applications for tissue histology and enrichment of RNA for gene expression using adjacent regions from a single mouse skeletal muscle. Typically, it is challenging to achieve adequate homogenization of small skeletal muscle samples because buffer volumes may be too low for efficient grinding applications, yet without sufficient mechanical disruption, the dense tissue architecture of muscle limits penetration of buffer reagents, ultimately causing low RNA yield. By following the protocol reported here, 30 &#956;m sections are collected and pooled allowing cryosectioning and subsequent needle homogenization to mechanically disrupt the muscle, increasing the surface area exposed for buffer penetration. The primary limitations of the technique are that it requires a cryostat, and it is relatively low throughput. However, high-quality RNA can be obtained from small samples of pooled muscle cryosections, making this method accessible for many different skeletal muscles and other tissues. Furthermore, this technique enables matched analyses (e.g., tissue histopathology and gene expression) from adjacent regions of a single skeletal muscle so that measurements can be directly compared across applications to reduce experimental uncertainty and to reduce replicative animal experiments necessary to source a small tissue for multiple applications.

Consecutive cryo-sections are collected to enable histological applications and enrichment of RNA for gene expression measurements using adjacent regions from a single mouse skeletal muscle. High-quality RNA is obtained from 20 - 30 mg of pooled cryosections and measurements are directly compared across applications.

유전자 발현 및 조직 학적 분석을위한 단일 마우스 골격근의 연속 지역의 냉동 절편

With this method, consecutive cryosections are collected to enable both microscopy applications for tissue histology and enrichment of RNA for gene ...

A Multi-hole Cryovial Eliminates Freezing Artifacts when Muscle Tissues are Directly Immersed in Liquid Nitrogen

Studies on skeletal muscle physiology face the technical challenge of appropriately processing the specimens to obtain sections with clearly visible cytoplasmic compartments. Another hurdle is the tight apposition of myofibers to the surrounding tissues. Because the process of tissue fixation and paraffin embedding leads to the shrinkage of muscle fibers, freezing is an optimal means of hardening muscle tissue for sectioning. However, a commonly encountered issue, the formation of ice crystals, occurs during the preparation of frozen sections because of the high water content of muscle. The protocol presented here first describes a simple and efficient method for properly freezing muscle tissues by immersing them in liquid nitrogen. The problem with using liquid nitrogen alone is that it causes the formation of a nitrogen gas barrier next to the tissue, which acts as an insulator and inhibits the cooling of the tissues. To avoid this "vapor blanket" effect, a new cryovial was designed to increase the speed of liquid flow around the tissue surface. This was achieved by punching a total of 14 inlet holes in the wall of the vial. According to bubble dynamics, a higher rate of liquid flow results in smaller bubbles and fewer chances to form a gas barrier. When liquid nitrogen flows into the cryovial through the inlet holes, the flow velocity around the tissue is fast enough to eliminate the gas barrier. Compared to the method of freezing muscle tissues using pre-chilled isopentane, this protocol is simpler and more efficient and can be used to freeze muscle in a throughput manner. Furthermore, this method is optimal for institutions that do not have access to isopentane, which is extremely flammable at room temperature.

This protocol describes a procedure to freeze muscle tissues by plunging them directly into liquid nitrogen. This protocol also highlights a new cryovial that can avoid the &#34;blanket effect&#34; of nitrogen gas when liquid nitrogen contacts the tissue surface of a specimen.

근육 조직이 직접 액체 질소에 몰입 할 때 이슈 결빙 멀티 홀 Cryovial 제거

Studies on skeletal muscle physiology face the technical challenge of appropriately processing the specimens to obtain sections with clearly visible ...

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal muscle development and regeneration, and the autophagic process has been described in several muscular pathologies and age-related muscle disorders. A recently described block of the autophagic process that correlates with the functional exhaustion of satellite cells during muscle repair supports the notion that active autophagy is coupled with productive muscle regeneration. These data uncover the crucial role of autophagy in satellite cell activation during muscle regeneration in both normal and pathological conditions, such as muscular dystrophies. Here, we provide a protocol to monitor the autophagic process in the adult Muscle Stem Cell (MuSC) compartment during muscle regenerative conditions. This protocol describes the setup methodology to perform in situ immunofluorescence imaging of LC3, an autophagy marker, and MyoD, a myogenic lineage marker, in muscle tissue sections from control and injured mice. The methodology reported allows for monitoring the autophagic process in one specific cell compartment, the MuSC compartment, which plays a central role in orchestrating muscle regeneration.

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Active autophagy is associated with productive muscle regeneration, which is essential for Muscle Stem Cell (MuSC) activation. Here, we provide a protocol for the in situ detection of LC3, an autophagy marker in MyoD-positive MuSCs of muscle tissue sections from control and injured mice.

근육 줄기 세포에서 Autophagy의 원위치 Immunofluorescent 얼룩

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal ...

Immunolabelling Myofiber Degeneration in Muscle Biopsies

The necrosis of muscle fibres (myonecrosis) plays a central role in the pathogenesis of several muscle conditions, including muscular dystrophies. Therapeutic options addressing the causes of muscular dystrophy pathogenesis are expected to alleviate muscle degeneration. Therefore, a method to assay and quantify the extent of cell death in muscle biopsies is needed. Conventional methods to observe myofiber degeneration in situ are either poorly quantitative or rely on the injection of vital dyes. In this article, an immunofluorescence protocol is described that stains necrotic myofibers by targeting immunoglobulin G (IgG) uptake by myofibers. The IgG uptake method is based on cell features characterizing the necrotic demise, including 1) the loss of plasma membrane integrity with the release of damage-associated molecular patterns and 2) the uptake of plasmatic proteins. In murine cross-sections, the co-immunolabelling of myofibers, extracellular matrix proteins, and mouse IgG allows clean and straightforward identification of myofibers with necrotic fate. This simple method is suitable for quantitative analysis and applicable to all species, including human samples, and does not require the injection of vital dye. The staining of necrotic myofibers by IgG uptake can also be paired with other co-immunolabelling.

Described here is a protocol for direct immunolabelling of necrotic myofibers in muscle cryosections. Necrotic cells are permeable to serum proteins, including immunoglobulin G (IgG). Revealing the uptake of IgG by myofibers allows the identification and quantification of myofibers that undergo necrosis regardless of muscle condition.