The authors would like to thank the Biomarker Imaging&#160;Research Laboratory (BIRL) at Sunnybrook Research Institute for their histology services. Support for this work was provided by the Terry Fox Foundation, the Canadian Breast Cancer Foundation&#8208;the Prairie&#8208;NWT as well as a CIHR grant, #MOP-97996.

1. 표본 <ol><li> 사흘 수유의 발병을 게시 야생형 CDH1에서 수술 유방 동맥뿐만 아니라 IGFBP7 널 마우스를 절제. </li><li> 기본 유선 형태를 회복하는 데 도움이 유리 슬라이드에 분비를 확산. </li></ol> 2. 고정 및 조직 처리 <ol><li> 4 O C.에서 중립 버퍼 4 % PFA O / N에있는 유방 동맥 수정 </li><li> 이전 조직 처리에 70 % 에탄올에 분비를 저장합니다. </li><li> 작은 조직 처리 카세트에 분비를 전송합니다. </li><li> 자동 조직 프로세서를 사용하여 조직을 처리 <ol><li> 45 분 자일 렌의 1 시간과 2 시간 동안 100 % 에탄올, 45 분 동안 95 %의 에탄올에 45 분, 2 회 3 회 70 % 에탄올 에탄올, 자일 렌 화장실을 증가하는 조직을 탈수. </li><li> 인가 압력이 진공에서 1 시간마다 파라핀 3 회에 조직을 침투. </li></ol></li><li> 블록을 형성하기 위해 파라핀의 조직을 포함, 절편. </li></ol> 3. 조직학 및 Blockface 영상 <ol><li> 과량의 파라핀이 제거 될 때까지 회전 마이크로톰을 사용하여 파라핀 블록을 트림. </li><li> 카세트에 수직 파라핀 블록의 적어도 두 모서리 1 ㎜의 구멍을 드릴 수직 밀링 기계를 사용한다. </li><li> 회전하는 마이크로톰의 조직 블록을 설치합니다. </li><li> 마이크로톰 앞에 blockface 이미징 시스템 (19)을 설정합니다. </li><li> 이전 절편을 광학 blockface 이미지를 캡처합니다. </li><li> 마이크로톰에 5 μm의 두께로 네 부분의 리본을 잘라. <ol><li> 차가운 물을 욕조에 리본을 전송합니다. </li><li> 리본의 제 2 및 제 4 섹션을 분리하고 현미경 슬라이드에 그들을 마운트합니다. 제 2 및 제 4 섹션을 선택하면 부분 사이에 5 μm의 간격을 제공합니다. </li><li> 는 현미경 슬라이드에 다시 장착 한 후, 그것을 unwrinkle에 따뜻한 물을 욕조 (48 오 C)의 각 섹션을 확장합니다. 참고 : C우팅,, 설치 섹션 같은 눈물, 배, 수축, 팽창 등의 부분에 약간의 왜곡을 일으킬 unwrinkling. 이러한 아티팩트는 조직학 섹션의 등록을 복잡. </li><li> 자동 염색기를 사용하여 H &amp; E와 섹션을 얼룩. </li><li> 자동 coverslipper를 사용하여 슬라이드를 커버 슬립. </li><li> 또한 해상도로 디지털 조직학 슬라이드 스캐너를 사용하여 슬라이드를 디지털화. 이 프로토콜에 대한 배율은 20 배이며, 해상도는 0.47 μm의 수 있습니다. </li><li> blockface 이미지, 18 μm의 해상도에 조직학 이미지를 다운 샘플링. </li></ol></li></ol> 4. 이미지 등록 <ol><li> 이미지 분할 포인트 선택 <ol><li> blockface의 이미지는 등록 구멍의 화소 값을 측정하고 세그먼트 파라핀 블록의 모서리에있는 등록의 홀에 고정 된 임계 값으로서 평균값을 사용한다. </li><li> 몇 가지 추가 부품도에 의해 분할 될 수 있기 때문에고정 된 임계 값을 사용하여, 원형도 및 정공을 찾아 여분 오브젝트를 폐기 세그먼트 객체의 영역을 사용한다. 이렇게 작은 코드를 작성하고 분할 된 개체에 대한 (4π X 면적) / (주변) 2의 비율을 찾을 수 있습니다. 둥근 물체에 대한이 비율은 1입니다. </li><li> 각 유선 들어, 참조로 한 blockface 콘텐츠를 선택하고 등록 구멍 및 경계표 기반 등록 기술의 중심을 이용하여 기준에 blockface 이미지의 나머지를 정렬. </li><li> 정렬 blockface의 이미지에 대해서는, 수동 세그먼트 또는 배경에서 조직을 추출합니다. 프로토콜의 나머지 부분에 대한 마스크의 대부분 상당한 크기의 개체를 사용합니다. </li><li> H &amp; E에 대한 부분은 자동 분할 아래의 단계를 따릅니다. <ol><li> 배경에서 세그먼트 이미지에 오쓰 임계 기술 (20)를 사용하고 조직학 이미지의 바이너리 마스크를 만들 수 있습니다. </li><li> 식별 및 시간을 사용하여 각 마스크에 가장 상당한 개체를 선택표시된 개체의 istogram. </li><li> 조직학 및 blockface 마스크 모두에서 폭이 1 픽셀 경계 지점의 압축을 풉니 다. </li><li> 조각 별 선형의 시퀀스에 의해 경계 지점을 나타내는, 사슬 코드 알고리즘 (21)을 사용하여 적합하다. </li></ol></li></ol></li><li> 초기 강성 등록 <ol><li> 초기 강성은 조직학의 경계 지점 및 해당 blockface 이미지 사이의 변환을 찾기 위해, 푸리에 기술자에게 알고리즘 (22)를 사용합니다. 이 초기 변환은 변환, 회전 및 배율 요소가 포함되어 있습니다. </li><li> 초기는 이전 단계에서 수득되는 변환 각각 조직학 콘텐츠 변환. </li></ol></li><li> 엄밀한 등록 제련 <ol><li> 롤링 볼 필터 (23)를 사용하여 조직 학적 윤곽에서 곡률의 가장자리 부분을 제거합니다. </li><li> 무작위로 균일 한 분포를 사용하여 나머지 조직 학적 경계 지점에서 500 포인트를 선택합니다. </li><li> 변환푸리에 설명에서 얻은 초기 변화와 조직 학적 임의의 경계 지점. </li><li> blockface 경계 지점의 전체 집합을 선택하고 blockface 경계 지점, 대상 및 조직학 임의의 경계 지점의 강성 변화를 찾기 위해 반복 가장 가까운 포인트 (ICP) 알고리즘 (24)를 사용합니다. </li><li> 정렬 조직학 이전 단계에서 얻은 이미지와 정렬 조직학 이미지의 스택 조직학 볼륨을 생성하고 변환 할 수 있습니다. </li><li> 조직학 볼륨의 시각적 이미지를 만드는 3D 시각화 소프트웨어를 사용합니다. </li></ol></li><li> 배의 배율로 이미지의 스택보기 <ol><li> 배율 5 배 원래 조직학 이미지를 다운 샘플링. </li><li> 조직학 이미지 중 하나에 관심 영역을 자릅니다. </li><li> 등록이 단계에서 강성 변환의 조합을 사용하여 다른 5X 조직학 이미지에 해당 영역의 위치를​​ 계산한다. </li><li> REGI 자르기다른 모든 조직 학적 이미지에 같은 크기의 영역으로 그 기능. </li><li> 마지막으로 수동으로 영역 사이의 정렬을 구체화합니다. 두 개의 이미지를 오버레이 및 회전과 다른 하나 이상의 이미지 중 하나의 번역에 대한 값을 선택할 수 있도록 한 다음 정렬이 허용됩니다 변환 된 이미지를 저장하는 프로그램을 작성하라. </li><li> 3D 시각화 소프트웨어를 사용하여 정렬 배 조직학 영역의 스택을 볼 수 있습니다. </li></ol></li></ol>

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The authors have nothing to disclose

이 연구에서, 우리는 조직을 왜곡 할 수있는 조직 내에서 내부 무작위로 선택된 랜드 마크 또는 이식 기준 마커를 필요로하지 않는 직렬 2D 조직학 이미지에서 3D 조직학 볼륨을 재구성하기 이미지 등록 워크 플로우를 개발했습니다. 기재된 방법에 의해, 광 blockface 이미지 자체는 종래 구획에 참조 화상으로서 사용된다. 우리는 blockface 이미지 정렬을 돕기 위해 및 카메라 앞에서 파라핀 블록의 2 차...

IGFBP7 (단백질 7 바인딩 성장 인자와 같은 인슐린)는 IGF-결합 단백질 가족의 일원이며, IGF1 수용체 4를 결합하기 위해 표시되었습니다. 이종 이식 종양 모델에서 IGFBP7의 재 도입이 크게 세포 사멸과 세포 노화 (7)의 유도를 통해 6 성장 종양을 억제하면서 IGFBP7의 하향 조정은, 유방암 5의 나쁜 예후와 상관 관계가있는 것으로 알려져있다. IGFPB7의 효과를 연구하기 위해, IGFBP7 null이 마우스는 5 (게시되지 않은 데이터)를 만들었습니다. 이 마우스가 종양을 개발하지 않는 동안, 난소, 근육과 간 조직 학적 변화뿐만 아니라 유선 발달 패턴 (게시되지 않은 데이터)의 결함을 보여줍니다. 널 마우스는 작은 쓰레기 크기가 여러 큰 새끼 (게시되지 않은 데이터)를 유지 할 수없는 등 결함이 표현형을 먼저 표시했다. 3D 조직학 볼륨 유용 INFORMAT을 제공하는 잠재력을 가지고부피 의료 영상의 병리 소견의 양적 비교 분석과 평가를 위해 이온. 입체 촛점, 이광자 현미경 지역 범위 (14)에서 동맥의 고해상도 세포 형태 학적 정보를 제공 할 수 있지만, 볼과 깊이의 제한된 필드를 갖는다. 조직학 볼륨 재구성이 훨씬 더 큰 공간 범위를 통해 자세한 정보를 제공합니다. 약간의 왜곡이 같은 수축, 팽창, 눈물, 그리고 주름 등의 조직 학적 섹션의 준비 기간 동안 예상되는 전통적인 접근 방법을 사용. 이러한 왜곡은 어려운 3D 볼륨을 재구성하는 3 차원 스택으로 직렬 조직학 이미지를 등록 할 수 있습니다. 결함 연속 섹션 수를 그대로 섹션 사이의 유사성을 증가함에 따라 감소하고 결과적 등록 프로세스는 더욱 복잡하게된다. 다른 방법은 조직 학적 섹션을 등록하고 연속 조직학 VO를 생성하는 것이 제안되어있다륨. 일부 기술은 강도의 변화 (8)에 따라, 그리고 다른 사람은 섹션 9의 모양을 기반으로합니다. 일부 시편 해부 구조는 랜드 기반 등록 방법 12,13와 함께 마크 (10, 11)로서 사용될 수있다. 그러나 이러한 내부 구조는 전체 볼륨에 걸쳐 더 신뢰할 수있는 해부학 적 구조를 식별 할 수있는 표본에 대해 감지하지 않을 수 있습니다. 일부 그룹은 짝 등록 방법을 사용하고 연속 조직학 이미지보기 윤곽 또는 해부학 적 구조를 16 ~ 18을 사용하여 다른 하나를 등록했습니다. 참조 이미지의 사용없이 서로 직렬 조직학 섹션 등록은 레지스트레이션 오차를 전파하고 조직학 볼륨의 실제 형상을 변경할 수있다. 쌍대 등록 방법은 이미지의 스택에 걸쳐 조직학 섹션 및 내부 구조물의 형상의 일관성에 의존한다; 그 때문에, 시료의 치밀 샘플링을 요구하는임상 검체에 대한 예를 들어, 수도 항상 가능한 것은 아니다. 이 파이프 라인에서 우리는 조직 학적 볼륨 재구성 19 참조 이미지의 집합으로 blockface 이미지를 사용합니다. Blockface 이미지는 마이크로톰에 장착 한 후 파라핀 조직 블록으로 촬영되며 각 섹션은 절단하기 전에. 따라서, 각각의 시리얼 섹션 컷 손상 시리얼 섹션 8,11,15의 등록을 방해하지 않습니다. 우리는 다른 그룹에서 다른 방식 blockface 이미지를 캡처. 광학 블록 얼​​굴 이미지는 광학 정기적으로 렌즈를 사용할 때 일반적으로 발생 배럴 및 관점 왜곡을 제거하거나 최소화하기 위해 텔레 센 트릭 렌즈에 의해 얻을 수있다. 이 레귤러 렌즈를 사용 blockface 촬상을 수행 게시 된 다른 방법을 통해 제안 된 방법의 장점 중 하나이다. 이미지는 담배 마는 간의 콘트라스트 개선을위한 블록의 표면의 반사를 사용하여 약간 비스듬한 각도에서 찍은UE와 파라핀 표면과 파라핀 표면 아래 깊이에서 조직의 그림자를 제거하는 방법. 사진 필터는 블록 표면 및 콘트라스트 19 균형 조직으로부터 나오는 광을 편광하기 위해 사용된다. 회전하는 마이크로톰에 블록의 변위를 교정하려면 2-3 구멍 blockface 이미지를 쉽게 감지 할 수있는 블록의 모서리에 드릴 수 있습니다. 이 구멍의 무게 중심은 blockface 이미지를 정렬하는 랜드 마크 기반의 엄격한 등록과 함께 사용된다.

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reconstruction of 3-dimensional histology volume and its application to study mouse mammary glands

조직학 볼륨 재구성 된 3D 형상 및 셀 이루어진 macrostructures의 수준에서 장기의 부피 변화의 연구를 용이하게한다. 또한 부피 의료 영상 및 치료에 새로운 기술과 알고리즘을 조사하고 유효성을 검사 할 수 있습니다. 다른 장기 1,2,3의 3D 고해상도지도 책 만들기 조직학 볼륨 재건의 또 다른 응용 프로그램입니다. 이 조직 구조 및 다양한 세포 기능 간의 공간 관계를 조사하기위한 자원을 제공한다. 우리는 광 blockface 이미지 세트를 사용 조직학 대량 재건을위한 이미지 등록 방식을 제시한다. 재구성 된 조직학 볼륨없이 전파되는 후 처리 등록 오류로 처리 된 시편의 신뢰할 수있는 모양을 나타냅니다. 두 개의 마우스 유방 땀샘의 hematoxylin 및 eosin (H &amp; E) 염색 부분은 ED에서 추출 된 경계 지점을 사용하여 해당 blockface 이미지에 등록 된조직학 및 blockface 이미지에서 시편의 GES. 등록의 정확성은 육안으로 평가 하였다. 유 방 땀 샘의 macrostructures의 정렬은 시각적으로 높은 해상도로 평가되었다. 이 연구는 유선의 절제에서 3D 조직학 볼륨 재구성을 통해 이르기까지,이 이미지 등록 파이프 라인의 여러 단계를 묘사한다. 2D 조직학 이미지 섹션 쌍 간의 구조적 차이점을 계시하면서, 3 차원 조직학 부피는 모양과 젖샘의 체적의 차이를 시각화 할 수있는 능력을 제공한다.

전통적인 기법 현미경 함정 미세한 수준에서 기관의 이해는 한번에 하나의 시야에 한정된다는 점이다. 심지어 전체 슬라이드 섹션을 제공 &quot;전체 개시&quot;슬라이드, 입체 정보를 제공하지 못한다. 전체 슬라이드, 동적 인 스캐닝 기술, 전체가 부분을 볼 수있는 우리의 능력이 증가의 발전 그러나 구조를 추정하는 것은 3D 조직학 볼륨 재구성이 필요합니다. 더 IGFBP7 널 마우...

Watch this Scientific Journal Video about Reconstruction of 3-Dimensional Histology Volume and its Application to Study Mouse Mammary Glands at JoVE.com

Reconstruction of 3-Dimensional Histology Volume and its Application to Study Mouse Mammary Glands

We present an image registration approach for 3-dimensional (3D) histology volume reconstruction, which facilitates the study of the changes of an organ at the level of macrostructures made up of cells . Using this approach, we studied the 3D changes between wild-type and Igfbp7-null mammary glands.

3 차원 조직학 볼륨 및 마우스 유방 땀샘을 연구에의 응용의 재구성

Histology volume reconstruction facilitates the study of 3D shape and volume change of an organ at the level of macrostructures made up of cells. It can ...

reconstruction-3-dimensional-histology-volume-its-application-to

Research

JoVE Journal

Biology

14.3K 조회수.  University of Toronto. We present an image registration approach for 3-dimensional (3D) histology volume reconstruction, which facilitates the study of the changes of an organ at the level of macrostructures made up of cells . Using this approach, we studied the 3D changes between wild-type and Igfbp7-null mammary glands. 

비디오: Reconstruction of 3-Dimensional Histology Volume and its Application to Study Mouse Mammary Glands

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

도마뱀의 운동의 속도와 방향에 시각 민감도 테스트

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

연구

생물학

The Ladder Rung Walking Task: A Scoring System and its Practical Application.

Progress in the development of animal models for/stroke, spinal cord injury, and other neurodegenerative disease requires tests of high sensitivity to elaborate distinct aspects of motor function and to determine even subtle loss of movement capacity. To enhance efficacy and resolution of testing, tests should permit qualitative and quantitative measures of motor function and be sensitive to changes in performance during recovery periods. The present study describes a new task to assess skilled walking in the rat to measure both forelimb and hindlimb function at the same time. Animals are required to walk along a horizontal ladder on which the spacing of the rungs is variable and is periodically changed. Changes in rung spacing prevent animals from learning the absolute and relative location of the rungs and so minimize the ability of the animals to compensate for impairments through learning. In addition, changing the spacing between the rungs allows the test to be used repeatedly in long-term studies. Methods are described for both quantitative and qualitative description of both fore- and hindlimb performance, including limb placing, stepping, co-ordination. Furthermore, use of compensatory strategies is indicated by missteps or compensatory steps in response to another limb&#x2019;s misplacement. 

The ladder rung walking task is a new test to assess skilled walking and measure both forelimb and hindlimb placing, stepping, and inter-limb co-ordination.

작업 워킹 레더 렁 : 채점 시스템과 실용적인 프로그램입니다.

Progress in the development of animal models for/stroke, spinal cord injury, and other neurodegenerative disease requires tests of high sensitivity to ...

Lens Transplantation in Zebrafish and its Application in the Analysis of Eye Mutants

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development can be addressed. The zebrafish is useful for genetic studies due to several advantageous characteristics, including small size, high fecundity, short lifecycle, and ease of care. Lens development occurs rapidly in zebrafish. By 72 hpf, the zebrafish lens is functionally mature [3]. Abundant genetic and molecular resources are available to support research in zebrafish. In addition, the similarity of the zebrafish eye to those of other vertebrates provides basis for its use as an excellent animal model of human defects[4-7]. Several zebrafish mutants exhibit lens abnormalities, including high levels of cell death, which in some cases leads to a complete degeneration of lens tissues [8]. To determine whether lens abnormalities are due to intrinsic causes or to defective interactions with the surrounding tissues, transplantation of a mutant lens into a wild-type eye is performed. Using fire-polished metal needles, mutant or wild-type lenses are carefully dissected from the donor animal, and transferred into the host. To distinguish wild-type and mutant tissues, a transgenic line is used as the donor. This line expresses membrane-bound GFP in all tissues, including the lens. This transplantation technique is an essential tool in the studies of zebrafish lens mutants.

Lens development involves interactions with other tissues. Several zebrafish eye mutants are characterized by an abnormally small lens size. Here we demonstrate a lens transplantation experiment to determine whether this phenotype is due to intrinsic causes or defective interactions with tissues that surround the lens.

렌즈 Zebrafish의 이식과 눈 돌연변이의 분석에의 응용

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development ...

Mouse Mammary Epithelial Cells form Mammospheres During Lactogenic Differentiation

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of dome shaped cell structures referred to as mammospheres 1. The HC11 cell line has been employed as a model system for the study of regulation of mammary lactogenic differentiation both in vitro and in vivo 2. The HC11 cells differentiate and synthesize milk proteins in response to treatment with lactogenic hormones. Following the growth of HC11 mouse mammary epithelial cells to confluence, lactogenic differentiation was induced by the addition of a combination of lactogenic hormones including dexamethasone, insulin, and prolactin, referred to as DIP. The HC11 cells induced to differentiate were photographed at times up to 120 hours post induction of differentiation and the number of mammospheres that appeared in each culture was enumerated. The size of the individual mammospheres correlates with the degree of differentiation and this is depicted in the images of the differentiating cells.

HC11 lactogenic differentiation can be characterized by the formation of domed structures referred to as mammospheres. The structures can be enumerated by phase contrast microscopy to aid in quantifying lactogenic differentiation.

마우스 내유 상피 세포는 Lactogenic 차별 동안 Mammospheres를 형성

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of ...

Evaluation of Mammary Gland Development and Function in Mouse Models

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves composed of a number of terminal duct lobular units made of secretory alveoli and converging ducts1. In mice, a similar architecture is observed at pregnancy in which ducts and alveoli are interspersed within the connective tissue stroma. The mouse mammary gland epithelium is a tree like system of ducts composed of two layers of cells, an inner layer of luminal cells surrounded by an outer layer of myoepithelial cells denoted by the confines of a basement membrane2. At birth, only a rudimental ductal tree is present, composed of a primary duct and 15-20 branches. Branch elongation and amplification start at the beginning of puberty, around 4 weeks old, under the influence of hormones3,4,5. At 10 weeks, most of the stroma is invaded by a complex system of ducts that will undergo cycles of branching and regression in each estrous cycle until pregnancy2. At the onset of pregnancy, a second phase of development begins, with the proliferation and differentiation of the epithelium to form grape-shaped milk secretory structures called alveoli6,7. Following parturition and throughout lactation, milk is produced by luminal secretory cells and stored within the lumen of alveoli. Oxytocin release, stimulated by a neural reflex induced by suckling of pups, induces synchronized contractions of the myoepithelial cells around the alveoli and along the ducts, allowing milk to be transported through the ducts to the nipple where it becomes available to the pups 8. Mammary gland development, differentiation and function are tightly orchestrated and require, not only interactions between the stroma and the epithelium, but also between myoepithelial and luminal cells within the epithelium9,10,11. Thereby, mutations in many genes implicated in these interactions may impair either ductal elongation during puberty or alveoli formation during early pregnancy, differentiation during late pregnancy and secretory activation leading to lactation12,13. In this article, we describe how to dissect mouse mammary glands and assess their development using whole mounts. We also demonstrate how to evaluate myoepithelial contractions and milk ejection using an ex-vivo oxytocin-based functional assay. The effect of a gene mutation on mammary gland development and function can thus be determined in situ by performing these two techniques in mutant and wild-type control mice.

This method describes how to dissect and assess mammary gland development and function from mice. Excised mammary glands are assessed for the degree of development using whole mount while milk ejection is evaluated using an oxytocin-based myoepithelial cell contraction assay.

마우스 모델에서 유선 개발 및 기능 평가

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves ...

Intraductal Injection for Localized Drug Delivery to the Mouse Mammary Gland

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of genes within the mammary epithelium has been difficult to achieve due to the lack of appropriate targeting molecules that are independent of developmental stages such as pregnancy and lactation. Herein, we describe a technique for localized delivery of reagents to the mammary gland at any stage in adulthood via intraductal injection into the nipples of mice. The injections can be performed on live mice, under anesthesia, and allow for a non-invasive and localized drug delivery to the mammary gland. Furthermore, the injections can be repeated over several months without damaging the nipple. Vital dyes such as Evans Blue are very helpful to learn the technique. Upon intraductal injection of the blue dye, the entire ductal tree becomes visible to the eye. Furthermore, fluorescently labeled reagents also allow for visualization and distribution within the mammary gland. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents, and small molecules.

A protocol for the non-invasive intraductal delivery of aqueous reagents to the mouse mammary gland is described. The method takes advantage of localized injection into the nipples of mammary glands targeting mammary ducts specifically. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents and small molecules.

마우스 유선 화 된 약물 전달을위한 췌관 주입

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of ...

A Fluorescence-based Exonuclease Assay to Characterize DmWRNexo, Orthologue of Human Progeroid WRN Exonuclease, and Its Application to Other Nucleases

WRN exonuclease is involved in resolving DNA damage that occurs either during DNA replication or following exposure to endogenous or exogenous genotoxins. It is likely to play a role in preventing accumulation of recombinogenic intermediates that would otherwise accumulate at transiently stalled replication forks, consistent with a hyper-recombinant phenotype of cells lacking WRN. In humans, the exonuclease domain comprises an N-terminal portion of a much larger protein that also possesses helicase activity, together with additional sites important for DNA and protein interaction. By contrast, in Drosophila, the exonuclease activity of WRN (DmWRNexo) is encoded by a distinct genetic locus from the presumptive helicase, allowing biochemical (and genetic) dissection of the role of the exonuclease activity in genome stability mechanisms. Here, we demonstrate a fluorescent method to determine WRN exonuclease activity using purified recombinant DmWRNexo and end-labeled fluorescent oligonucleotides. This system allows greater reproducibility than radioactive assays as the substrate oligonucleotides remain stable for months, and provides a safer and relatively rapid method for detailed analysis of nuclease activity, permitting determination of nuclease polarity, processivity, and substrate preferences.

Exonucleases play critical roles in ensuring genome stability. Loss of WRN exonuclease function results in premature aging. Studying substrates and other requirements of the nuclease in vitro can help elucidate its role in vivo. Here we demonstrate a rapid and reproducible fluorescence-based assay to measure its nuclease activity.

WRN exonuclease is involved in resolving DNA damage that occurs either during DNA replication or following exposure to endogenous or exogenous genotoxins. ...

Application of Retinoic Acid to Obtain Osteocytes Cultures from Primary Mouse Osteoblasts

The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell numbers. Therefore, the most widely used cellular system is the osteocyte-like MLO-Y4 cell line.
The method here described refers to the use of retinoic acid to generate a homogeneous population of ramified cells with morphological and molecular osteocyte features.
After isolation of osteoblasts from mouse calvaria, all-trans retinoic acid (ATRA) is added to cell medium, and cell monitoring is conducted daily under an inverted microscope. First morphological changes are detectable after 2 days of treatment and differentiation is generally complete in 5 days, with progressive development of dendrites, loss of the ability to produce extracellular matrix, down-regulation of osteoblast markers and up-regulation of osteocyte-specific molecules.
Daily cell monitoring is needed because of the inherent variability of primary cells, and the protocol can be adapted with minimal variation to cells obtained from different mouse strains and applied to transgenic models. 
The method is easy to perform and does not require special instrumentation, it is highly reproducible, and rapidly generates a mature osteocyte population in complete absence of extracellular matrix, allowing the use of these cells for unlimited biological applications.

Treatment of primary mouse osteoblasts with retinoic acid produces a homogeneous population of ramified cells bearing morphological and molecular features of osteocytes. The method overcomes the difficulty of obtaining and maintaining primary osteocytes in culture, and can be advantageous to study cells derived from transgenic models.&#160;

기본 마우스 조골 세포에서 골 세포 배양을 구하는 레티노 산의 적용

The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell ...

High-throughput Image Analysis of Tumor Spheroids: A User-friendly Software Application to Measure the Size of Spheroids Automatically and Accurately

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to large-scale screening formats in every step of a drug screen, including large-scale image analysis. Currently there is no ready-to-use and free image analysis software to meet this large-scale format. Most existing methods involve manually drawing the length and width of the imaged 3D spheroids, which is a tedious and time-consuming process. This study presents a high-throughput image analysis software application &#8211; SpheroidSizer, which measures the major and minor axial length of the imaged 3D tumor spheroids automatically and accurately; calculates the volume of each individual 3D tumor spheroid; then outputs the results in two different forms in spreadsheets for easy manipulations in the subsequent data analysis. The main advantage of this software is its powerful image analysis application that is adapted for large numbers of images. It provides high-throughput computation and quality-control workflow. The estimated time to process 1,000 images is about 15 min&#160;on a minimally configured laptop, or around 1 min&#160;on a multi-core performance workstation. The graphical user interface (GUI) is also designed for easy quality control, and users can manually override the computer results. The key method used in this software is adapted from the active contour algorithm, also known as Snakes, which is especially suitable for images with uneven illumination and noisy background that often plagues automated imaging processing in high-throughput screens. The complimentary &#8220;Manual Initialize&#8221; and &#8220;Hand Draw&#8221; tools provide the flexibility to SpheroidSizer in dealing with various types of spheroids and diverse quality images. This high-throughput image analysis software remarkably reduces labor and speeds up the analysis process. Implementing this software is beneficial for 3D tumor spheroids to become a routine in vitro model for drug screens in industry and academia.

We present a high-throughput image analysis software application to measure the size of three-dimensional tumor spheroids imaged with bright-field microscopy. This application provides a fast and effective way to examine the effects of therapeutic drugs on spheroids, which is beneficial for researchers who wish to use spheroids in drug screens.

종양 상체의 높은 처리량의 이미지 분석 : 자동으로 정확하게 상체의 크기를 측정하는 사​​용자 친화적 인 소프트웨어 응용 프로그램

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to ...

Measuring Pressure Volume Loops in the Mouse

Understanding the causes and progression of heart disease presents a significant challenge to the biomedical community. The genetic flexibility of the mouse provides great potential to explore cardiac function at the molecular level. The mouse&#39;s small size does present some challenges in regards to performing detailed cardiac phenotyping. Miniaturization and other advancements in technology have made many methods of cardiac assessment possible in the mouse. Of these, the simultaneous collection of pressure and volume data provides a detailed picture of cardiac function that is not available through any other modality. Here a detailed procedure for the collection of pressure-volume loop data is described. Included is a discussion of the principles underlying the measurements and the potential sources of error. Anesthetic management and surgical approaches are discussed in great detail as they are both critical to obtaining high quality hemodynamic measurements. The principles of hemodynamic protocol development and relevant aspects of data analysis are also addressed.

This manuscript describes a detailed protocol for the collection of pressure-volume data from the mouse.

마우스에 압력 볼륨 루프 측정

Understanding the causes and progression of heart disease presents a significant challenge to the biomedical community. The genetic flexibility of the ...