NCI (U54 CA00100970), NCI (1 R03 CA150040 - 01), ACS (116602 - PF - 09 - 018-01 - 척수)

1. 복부 유선의 제거 및 전체 마운트 준비 <ol><li> IACUC 지침에 따라 동물을 안락사. 표면 보드에 바늘, 또는 테이프 다리의 다리로부터 뒷면에있는 동물을 핀, 그리고 에탄올 (EtOH)와 젖은 피부를 닦아냅니다. 피부 포셉와 약간을 들어 날카로운 가위가 목쪽으로 한 쌍의 # 5 젖꼭지 컷 사이에 시작하는 피부에 정중선 절개를 함께. 뒷다리쪽으로 정중선에서 거꾸로 Y 자 절개를합니다. 흉부 내유 분비가 수집하는 것도있다면, # 2 유두 앞 다리를 향해 쌍 사이의 정중선에서 또한 Y 자 절개를합니다. </li><li> 피부가 복부 # 4 유선을 폭로하기 위해 거꾸로 Y 절개의 한쪽에 가위로 오픈 절개하다, 내유 분비는 피부에 붙어있다. 완전히 선을 노출, 표면 보드에 바늘로 피부를 핀. 한 번에 하나의 측면과 선 (腺)을 작동합니다. </li><li> 가까운 유두로 근위 지역에서 시작과 동물의 척추쪽으로 선 (腺)의 말초 끝 부분 작업, 날카로운 가위 및 / 또는 메스를 사용 중 피부에서 무료로 유선을 해부하다. (이것은 특히 기존의 밀고로, 몇 분 정도 걸릴 수 있습니다.) </li><li> 즉시 적절하게 레이블이 붙은 유리 슬라이드 (적절한 영구 마커 펜 및 / 또는 &#39;다이아몬드 펜&#39;을 사용)로 분리 선 (腺)을 전파하고, 원래의 크기와 현장의 모양 신중하게 대응하는 선 (腺)을 전파. 유리 슬라이드는 선보다 더해야합니다. 슬라이드에 선 (腺)을 확산하면, 그것은 선 (腺)이 슬라이드에 막대기 있도록 잠시 동안 테이블에 앉아 보자,하지만 건조하게하지 않습니다. </li><li> Carnoy의 정착액 (75 % 빙초산, 25 % 에탄올을 절대 EtOH =)가 들어있는 병에 덮개를 씌우고, 그것이 2 일 이상 연기 후드의 실내 온도 (RT)에 고정하자. 슬라이드도 시간이 긴 기간 동안 정착액에 남아있을 수 있습니다. </li><li> RT에서 1 시간 동안 70 % EtOH에서 슬라이드를 씻으십시오. </li><li> RT 30 분 증류수로 씻어. </li><li> 슬라이드의 뒷면을 살펴 보길), (당신은 림프절이 통해 스테인드 것을 볼 때까지 이일 이상) *를 얼룩 카르민 알룸에 얼룩. </li><li> EtOH의 증가 시리즈, 각 1 시간에 세척 : 70 % -&gt; 95% -&gt; 100 %. 절대 EtOH의 마지막 세척 후 크실렌의 분비를 넣어. 최소한 2 일 RT에서 퓸 후드에 앉아 보자. 이 마지막 단계는 내유 지방 패드의 유선의 의미 delipidation의 삭제, 그리고 투명성에 후속 증가합니다. fattier 선은 더 이상 비운 시간이 필요입니다. </li><li> 같은 Permount로 설치 미디어를 사용하여 커버 - 전표로 마운트합니다. 스테레오 현미경으로 관찰하기 전에 (몇 일) 잘 슬라이드가 건조 보자. * 카르민 알룸는 얼룩이 : 플레이스 1g 카민 (시그마 C1022) 20 분 500 ML 증류수와 끓여서 2.5 g 알루미늄 칼륨 황산 (시그마 A7167). 물 500 ML에 마지막으로 볼륨을 조정합니다. 필터 및 냉동. 솔루션은 몇 달 동안 사용할 수 있습니다. 색상이 약한되면 폐기하십시오. </li></ol> 2. 유선 전체 마운트 형태론 분석 유선의 평생 마운츠 형태는 다음과 같은 엔드 - 포인트와 유방암 위험과 상관 결과에 따라 분석됩니다. <ol><li> 유선 쿨러 21 전체 마운트 (미리 pubertal 나이) : <ul><li> 상피 성장 : <ul><li> 유두에서 상피 나무의 끝 부분 (mm)로 거리는 통치자를 사용하여 측정 </li></ul></li><li> 악성 변화 가능성 : <ul><li> TEBs 수 (터미널 최종 꽃봉오리), 가벼운 현미경시켰다. TEBs는 내유 상피 나무의 말초 끝에있는 큰 주먹코 구조 아르 </li></ul></li><li> 차별 : <ul><li> AB1 (폐포 버드) 점수 (0-5). 폐포 꽃봉오리는 상피를 통해 확산됩니다. </li><li> AB2 점수 (0-5) </li></ul></li></ul> 홀스의 탑재는 가벼운 현미경 점수입니다. AB1 AB2과에서 점수 값은 최종 차별화 점수에 대한 추가됩니다. </li><li> 유선은 전체 장착 쿨러 50 (사후 pubertal 세)에서 수집된 : <ul><li> 상피 성장 : <ul><li> 림프절에서 상피 트리의 끝을 (mm)로 거리는 통치자를 사용하여 측정. </li><li> 상피 나무의 끝에서 지방 패드의 끝에 거리는 통치자를 사용하여 측정. </li></ul></li><li> 악성 변화 가능성 : <ul><li> 가벼운 현미경으로 계산 TEBs 수, </li></ul></li><li> 차별 : <ul><li> AB1 점수 (0-5) </li><li> AB2 점수 (0-5) </li><li> Lobules 점수 (0-5) </li></ul></li></ul> 홀스의 탑재는 가벼운 현미경 점수입니다. 차별은 두 가지 방법으로 평가됩니다. 첫째, AB1, AB2 및 lobules에서 점수 값은 최종 차별화 점수에 대한 추가됩니다. 또한, lobules 점수와 AB1 + AB2 점수 사이의 비율이 계산됩니다. ABS가 lobules로 구분하고, lobules와 ABS의 높은 비율, 더 차별화된 선 (腺)입니다. </li></ol> 3. 촉진과 고양이와 유선의 형태론에 상관 관계에 내유 종양 측정 <ol><li> 이 절차를 시작하려면 머리 뒤쪽 집게손가락과 반대 겨드랑이에서 엄지와 둘째 손가락으로, 다시 위로 손바닥으로 몸 전체를 쥐고하여 쥐를 잡아. 그것은 사후에 누워되도록 밀고 돌려, 및 내유 종양을 탐지하는 만져보다, 만져서 알 수있는 종양은 &quot;혹&quot;같은 느낌한다. </li><li> 다음 너비, 길이, 그리고 종양의 높이를 측정하는 캘리퍼스를 사용합니다. 타원체 공식 볼륨에게 = 1/6πabc, &#39;A&#39;= 폭, &#39;B&#39;= 길이, 그리고 &#39;C&#39;= 높이.를 사용하여 종양 볼륨을 계산 </li><li> 수동으로 주 단위로 노트북에있는 종양의 위치와 크기를 기록합니다. 이 데이터는 나중에 스프레드 시트로 전송됩니다. </li></ol> 4. 대표 결과 내유 지방 패드와 wholemount하기 위해 처리주의 절개는 유선의 발달 상태를 평가하실 수 있습니다. 각 단계가 올바르게 완료되면 전체 내유 상피 나무는 지방 패드 내에 명확하게 볼 수 있으며, 이것은 TEBs 및 폐포 꽃봉오리와 lobules의 밀도의 계산의 수를 쉽게 결정 수 있습니다. 준비 wholemounts에 잘못하면는 전체 지방 패드, 불충 분한 고정, 그리고 불충 분한 삭제를 해부하다하는 실패를 포함할 수 있습니다. 유선의 형태의 평가는 내유 종양 (TEBs), 상피 구조 (폐포의 꽃봉오리와 lobules)의 분화 정도, 그리고 성장의 측정 (ductal 신장)에 상승을 줄 수 구조의 현재의 수에 대한 정보를 제공합니다. 터미널 엔드에 TEBs을 구분하는 것이 중요하고, 후자는 마찬가지로 TEBs하는 상피의 말초 끝에있는,하지만 그들은 TEBs보다 작은 있으며, 악성 종양을 일으키다 아닙니다. 터미널 종료 또한 상피 트리 내에서 볼 수 있습니다. <img src="/files/ftp_upload/2260/2260fig1.jpg" alt="그림 1" /> 그림 1 :. 잘 준비 유선의 전체 탑재 대표 결과는 선 (腺)의 모든 구성 요소를 제대로 해부되어 있고 카르민 얼룩이 최적입니다. <img src="/files/ftp_upload/2260/2260fig2.jpg" alt="그림 2" /> 그림 2 :. 제대로 준비 유선의 전체 탑재 대표 결과 글랜드가 제대로 (선의 말초 부분이 누락)를 해부했습니다 제대로 뻗어되지 않았습니다 및 delipidation가 완전히 발생하지 않았습니다.

<ol>
	<li>Rasmussen, S. B., Young, L. J. T., Smith, G. H., Ip, M. M., Asch, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparing+mammary+gland+whole+mounts+form+mice.">Preparing mammary gland whole mounts form mice.</a> Methods in Mammary Gland Biology and Breast Cancer Research. , 75-85 (2000).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consensus+statements+from+Mammary+Gland+Whole+Mount+Round+Robin+meeting.">Consensus statements from Mammary Gland Whole Mount Round Robin meeting.</a> , (2009).</li><li>Hilakivi-Clarke, L., Clarke, R., Onojafe, I., Raygada, M., Cho, E., &amp;amp, L. i. p. p. m. a. n., E, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+maternal+diet+high+in+n-6+polyunsaturated+fats+alters+mammary+gland+development,+puberty+onset,+and+breast+cancer+risk+among+female+rat+offspring.">A maternal diet high in n-6 polyunsaturated fats alters mammary gland development, puberty onset, and breast cancer risk among female rat offspring.</a> Proc Natl Acad Sci. 94, 9372-9377 (1997).</li><li>Russo, I. H., Russo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammary+gland+neoplasia+in+long-term+rodent+studies.">Mammary gland neoplasia in long-term rodent studies.</a> Environ. Health Perspect. 104 (9), 938-967 (1996).</li><li>Hilakivi-Clarke, L., de Assis, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fetal+origins+of+breast+cancer.">Fetal origins of breast cancer.</a> TRENDS in Endocrinol and Metabol. 17 (9), 340-348 (2006).</li><li>Hilakivi-Clarke, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nutritional+modulation+of+terminal+end+buds:+its+relevance+to+breast+cancer+prevention.">Nutritional modulation of terminal end buds: its relevance to breast cancer prevention.</a> Curr Cancer Drug Targets. 7 (4), 465-474 (2007).</li><li>Silva Idos, S., De Stavola, B., Mccormack, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collaborative+Group+On+Pre-Natal+Risk+Factors+And+Subsequent+Risk+Of+Breast+Cancer.+Birth+size+and+breast+cancer+risk:+re-analysis+of+individual+participant+data+from+32+studies.">Collaborative Group On Pre-Natal Risk Factors And Subsequent Risk Of Breast Cancer. Birth size and breast cancer risk: re-analysis of individual participant data from 32 studies.</a> PloS Medicine. 5, e193-e193 (2008).</li><li>Warri, A., Saarinen, N. M., Makela, S., Hilakivi-Clarke, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+early+life+genistein+exposures+in+modifying+breast+cancer+risk.">The role of early life genistein exposures in modifying breast cancer risk.</a> Br J Cancer. 98, 1485-1493 (2008).</li><li>Russo, J., Hu, Y. F., Yang, X., Russo, I. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental,+cellular,+and+molecular+basis+of+human+breast+cancer.">Developmental, cellular, and molecular basis of human breast cancer.</a> J Natl Cancer Inst Monogr. , 17-37 (2000).</li><li>Oza, A. M., Boyd, N. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammographic+parenchymal+patterns:+A+marker+of+breast+cancer+risk.">Mammographic parenchymal patterns: A marker of breast cancer risk.</a> Epidemiological Reviews. 15, 196-208 (1993).</li></ol>

관심 없음 충돌 선언하지 않습니다.

유선 형태학의 최종 지점과 상피 나무의 성장을 평가 일찍 삶 식단 조작, 또는 utero 또는 prepubertal 호르몬 환경에서 변경 기타 조작은 나중에 유방암 위험을 수정 여부를 예측하는 데 사용될 수 있습니다. 인간의 유방암이 쥐에 TEBs와 비슷한 내유 구조 (터미널 ductal 소엽 단위, TDLUs)에서 시작되기 때문에,이 기법은 유방암 위험에 영향을 조기 삶의 노출의 가능성을 결정하는 데 사용할 수 있습니다. 또한, 여성의 높은 mammographic 밀도는 4-6 배로 유방암의 위험을 증가 시키며, 내유 상피 나무의 성장 평가가 mammographic 밀도이 밀도를 감소 요인을 결정하는 요인을 식별하는 데 사용할 수 있습니다. 이 정보를 얻으려면 유선이 완전히 해부를 제대로 모든 구성 요소가 현미경 시각 수 있도록 유리 슬라이드에 뻗어 있어야합니다. 또한, 적절한 카르민 얼룩과 크실렌의 delipidation은 유방암 위험 평가에 적합 전체 마운트를 제공합니다.

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
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		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Glacial acetic acid</td>
<td>&nbsp;</td>
<td>EM</td>
<td>AX0073-9</td>
<td>Carnoy's Fixative</td>
</tr>
<tr>
<td>Absolute ethanol</td>
<td>&nbsp;</td>
<td>The Waner-Graham Co.</td>
<td>64-17-5</td>
<td>Carnoy's Fixative</td>
</tr>
<tr>
<td>Carmine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C1022</td>
<td>Carmine solution</td>
</tr>
<tr>
<td>Aluminum potassium sulfate</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A7167</td>
<td>Carmine solution</td>
</tr>		</tbody>
		</table>
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changes in mammary gland morphology and breast cancer risk in rats

utero 및 pubertal 기간에 동안식이 / 호르몬 요인에 노출은 유선은 광범위한 모델링 및 다시 모델링을 거쳐 때 발암 물질 유발 내유 종양을 느끼기 쉬운 상태를 변경되는 유방암 쇼의 쥐 모델에서 연구. 비슷한 연구 결과는 인간에서 설명되었습니다 : 예를 들면, 높은 birthweight 유방암을 개발하고 이후 리스크 증가, 그리고 어린 시절 동안 간장의식이 섭취가 유방암 위험을 감소시킵니다. 그것은 이러한 태아와 출생 후의식이 수정이 차례로 나중에 유방암 위험을 수정할 것을 유선으로 영구 형태학의 변화를 유도 생각이다. 이러한 형태학의 변경 가능성이 같은 DNA의 methylation의 변화, histones하고 유전자 전사에 영향을 miRNA 표현으로 epigenetic 수정을 반영합니다. 이 문서에서는 유선의 형태에 변화가 쥐의 유방암 위험을 예측할 수있는 방법에 대해 설명합니다. 우리 프로토콜은 특별히 해부하다하고 제거 쥐의 복부 유선과 방법을 유선 전체 마운트를 준비하는 방법에 대해 설명합니다. 또한 세 엔드 포인트 (터미널 최종 꽃봉오리의 번호, 상피 연신율과 차별화)과 유방암을 개발의 위험을 예측하기 위해 데이터를 사용에 따라 유선의 형태를 분석하는 방법에 대해 설명합니다.

Watch this Scientific Journal Video about Changes in Mammary Gland Morphology and Breast Cancer Risk in Rats at JoVE.com

Changes in Mammary Gland Morphology and Breast Cancer Risk in Rats

우리 프로토콜은 쥐 복부 유선을 해부하다 방법과 유선 전체 마운트를 준비하는 방법에 대해 설명합니다. 또한 세 최종 포인트 (터미널 최종 꽃봉오리의 번호, 상피 연신율과 차별화)을 사용하여 유선의 형태를 분석하고식이 수정에 노출된 쥐의 유방암 위험을 예측하는 이러한 결과를 사용하는 방법에 대해 설명합니다<em&gt; utero에</em&gt; 또는 prepuberty 중.

쥐에서 유선의 형태론과 유방암 위험의 변화

Studies in rodent models of breast cancer show that exposures to dietary/hormonal factors during the in utero and pubertal periods, when the mammary gland ...

changes-in-mammary-gland-morphology-and-breast-cancer-risk-in-rats

Research

JoVE Journal

Biology

29.3K 조회수.  Georgetown University. 우리 프로토콜은 쥐 복부 유선을 해부하다 방법과 유선 전체 마운트를 준비하는 방법에 대해 설명합니다. 또한 세 최종 포인트 (터미널 최종 꽃봉오리의 번호, 상피 연신율과 차별화)을 사용하여 유선의 형태를 분석하고식이 수정에 노출된 쥐의 유방암 위험을 예측하는 이러한 결과를 사용하는 방법에 대해 설명합니다 utero에 또는 prepuberty 중.

비디오: Changes in Mammary Gland Morphology and Breast Cancer Risk in Rats

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 ...

연구

생물학

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts

Cellular invasion into local tissues is a process important in development and homeostasis. Malregulated invasion and subsequent cell movement is characteristic of multiple pathological processes, including inflammation, cardiovascular disease and tumor cell metastasis1. Focalized proteolytic degradation of extracellular matrix (ECM) components in the epithelial or endothelial basement membrane is a critical step in initiating cellular invasion. In tumor cells, extensive in vitro analysis has determined that ECM degradation is accomplished by ventral actin-rich membrane protrusive structures termed invadopodia2,3. Invadopodia form in close apposition to the ECM, where they moderate ECM breakdown through the action of matrix metalloproteinases (MMPs). The ability of tumor cells to form invadopodia directly correlates with the ability to invade into local stroma and associated vascular components3. 
Visualization of invadopodia-mediated ECM degradation of cells by fluorescent microscopy using dye-labeled matrix proteins coated onto glass coverslips has emerged as the most prevalent technique for evaluating the degree of matrix proteolysis and cellular invasive potential4,5. Here we describe a version of the standard method for generating fluorescently-labeled glass coverslips utilizing a commercially available Oregon Green-488 gelatin conjugate. This method is easily scaled to rapidly produce large numbers of coated coverslips. We show some of the common microscopic artifacts that are often encountered during this procedure and how these can be avoided. Finally, we describe standardized methods using readily available computer software to allow quantification of labeled gelatin matrix degradation mediated by individual cells and by entire cellular populations. The described procedures provide the ability to accurately and reproducibly monitor invadopodia activity, and can also serve as a platform for evaluating the efficacy of modulating protein expression or testing of anti-invasive compounds on extracellular matrix degradation in single and multicellular settings.

We describe the prototypical method for producing microscope coverslips coated with fluorescent gelatin for visualizing invadopodia-mediated matrix degradation. Computational techniques using available software are presented for quantifying the resultant levels of matrix proteolysis by single cells within a mixed population and for multicellular groups encompassing entire microscopic fields.

단일 및 다세포 상황에서 Invadopodia로 인한 세포 외 기질 Proteolysis의 양적 측정

Cellular invasion into local tissues is a process important in development and homeostasis.  Malregulated invasion and subsequent cell movement is ...

An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for inclusion and exclusion of data points, and computer-aided pattern recognition to support the interpretation and findings of the analysis. It has become increasingly important to be able to measure fluorescence images constructed from three-dimensional (3D) datasets in order to be able to capture the complexity of cellular dynamics and understand the basis of cellular plasticity within biological systems. Sophisticated microscopy instruments have permitted the visualization of 3D fluorescence images through the acquisition of multispectral fluorescence images and powerful analytical software that reconstructs the images from confocal stacks that then provide a 3D representation of the collected 2D images. Advanced design-based stereology methods have progressed from the approximation and assumptions of the original model-based stereology1 even in complex tissue sections2. Despite these scientific advances in microscopy, a need remains for an automated analytic method that fully exploits the intrinsic 3D data to allow for the analysis and quantification of the complex changes in cell morphology, protein localization and receptor trafficking. 
Current techniques available to quantify fluorescence images include Meta-Morph (Molecular Devices, Sunnyvale, CA) and Image J (NIH) which provide manual analysis. Imaris (Andor Technology, Belfast, Northern Ireland) software provides the feature MeasurementPro, which allows the manual creation of measurement points that can be placed in a volume image or drawn on a series of 2D slices to create a 3D object. This method is useful for single-click point measurements to measure a line distance between two objects or to create a polygon that encloses a region of interest, but it is difficult to apply to complex cellular network structures. Filament Tracer (Andor) allows automatic detection of the 3D neuronal filament-like however, this module has been developed to measure defined structures such as neurons, which are comprised of dendrites, axons and spines (tree-like structure). This module has been ingeniously utilized to make morphological measurements to non-neuronal cells3, however, the output data provide information of an extended cellular network by using a software that depends on a defined cell shape rather than being an amorphous-shaped cellular model. To overcome the issue of analyzing amorphous-shaped cells and making the software more suitable to a biological application, Imaris developed Imaris Cell. This was a scientific project with the Eidgen&ouml;ssische Technische Hochschule, which has been developed to calculate the relationship between cells and organelles. While the software enables the detection of biological constraints, by forcing one nucleus per cell and using cell membranes to segment cells, it cannot be utilized to analyze fluorescence data that are not continuous because ideally it builds cell surface without void spaces. To our knowledge, at present no user-modifiable automated approach that provides morphometric information from 3D fluorescence images has been developed that achieves cellular spatial information of an undefined shape (Figure 1). 
We have developed an analytical platform using the Imaris core software module and Imaris XT interfaced to MATLAB (Mat Works, Inc.). These tools allow the 3D measurement of cells without a pre-defined shape and with inconsistent fluorescence network components. Furthermore, this method will allow researchers who have extended expertise in biological systems, but not familiarity to computer applications, to perform quantification of morphological changes in cell dynamics.

We developed a software platform that utilizes Imaris Neuroscience, ImarisXT and MATLAB to measure the changes in morphology of an undefined shape taken from three-dimensional confocal fluorescence of single cells. This novel approach can be used to quantify changes in cell shape following receptor activation and therefore represents a possible additional tool for drug discovery.

3 차원 형광 이미지에서 세포 형태학의 변경 사항 단정 지을 분석 도구

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for ...

Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization

Fluorescent in situ hybridization using DNA probes on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA FISH) represents the most direct way to visualize the location of gene loci, chromosomal sub-regions or entire territories in individual cells. This type of analysis provides insight into the global architecture of the nucleus as well as the behavior of specific genomic loci and regions within the nuclear space. Immunofluorescence, on the other hand, permits the detection of nuclear proteins (modified histones, histone variants and modifiers, transcription machinery and factors, nuclear sub-compartments, etc). The major challenge in combining immunofluorescence and 3D DNA FISH is, on the one hand to preserve the epitope detected by the antibody as well as the 3D architecture of the nucleus, and on the other hand, to allow the penetration of the DNA probe to detect gene loci or chromosome territories 1-5. Here we provide a protocol that combines visualization of chromatin modifications with genomic loci in 3D preserved nuclei.

Here we describe a protocol for simultaneous detection of histone modifications by immunofluorescence and DNA sequences by DNA FISH followed by 3D microscopy and analyses (3D immuno-DNA FISH).

3D 원자력기구의 변화를 공부하기 위해 3D 보존 된 계면 핵에 결합 Immunofluorescence와 DNA의 물고기

Fluorescent in situ hybridization using DNA probes  on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA  FISH) represents the ...

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 ...

Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland

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

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

마우스 유선의 냉동 섹션에 간접 면역 형광

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

Comparison of Three Different Methods for Determining Cell Proliferation in Breast Cancer Cell Lines

Measuring cell proliferation can be performed by a number of different methods, each with varying levels of sensitivity, reproducibility and compatibility with high-throughput formatting. This protocol describes the use of three different methods for measuring cell proliferation in vitro including conventional hemocytometer counting chamber, a luminescence-based assay that utilizes the change in the metabolic activity of viable cells as a measure of the relative number of cells, and a multi-mode cell imager that measures cell number using a counting algorithm. Each method presents its own advantages and disadvantages for the measurement of cell proliferation, including time, cost and high-throughput compatibility. This protocol demonstrates that each method could accurately measure cell proliferation over time, and was sensitive to detect growth at differing cellular densities. Additionally, measurement of cell proliferation using a cell imager was able to provide further information such as morphology, confluence and allowed for a continual monitoring of cell proliferation over time. In conclusion, each method is capable of measuring cell proliferation, but the chosen method is user-dependent.

This protocol describes the use of three different methods for analyzing cell proliferation in breast cancer cell lines. This includes the use of conventional cell counting, luminescence-based cell viability, and cell counting through the use of a cell imager. Each offers advantages for the reproducible measurement of cell proliferation.

유방암 세포주에서 세포 증식을 결정하기위한 세 가지 방법 비교

Measuring cell proliferation can be performed by a number of different methods, each with varying levels of sensitivity, reproducibility and compatibility ...

Isolation of Intact, Whole Mouse Mammary Glands for Analysis of Extracellular Matrix Expression and Gland Morphology

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal architecture. Here, a small pocket below the skin was created using Mayo scissors, allowing separation of the glands within the subcutaneous tissue from the underlying peritoneum. Visualization of the glands was aided by the use of 3.5x-R surgical micro loupes. The pelt was inverted and pinned back allowing identification of the intact mammary fat pads. Each of the #4 abdominal glands was bluntly dissected by sliding the scalpel blade laterally between the subcutaneous layer and the glands. Immediately post-harvest, glands were placed in 10% neutral buffered formalin for subsequent tissue processing. Excision of the entire gland is advantageous because it primarily eliminates the risk of excluding important tissue-wide interactions between ductal epithelial cells and other microenvironmental cellular populations that could be missed in a partial biopsy. One drawback of the methodology is the use of serial sections from fixed tissues which limits analyses of ductal morphogenesis and protein expression to discrete locations within the gland. As such, changes in ductal architecture and protein expression in 3 dimensions (3D) is not readily obtainable. Overall, the technique is applicable to studies requiring whole intact murine mammary glands for downstream investigations such as developmental ductal morphogenesis or breast cancer.

Here, we present a protocol for the isolation of whole, intact mouse mammary glands to investigate extracellular matrix (ECM) expression and ductal morphology. Mouse #4 abdominal glands were extracted from 8-10 week old female nulliparous mice, fixed in neutral buffered formalin, sectioned and stained using immunohistochemistry for ECM proteins.

세포 외 기질 식 및 선 형태 분석을 그대로, 전체 마우스 유 방 땀 샘의 절연

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal ...

Bovine Mammary Gland Biopsy Techniques

Bovine mammary gland biopsies allow researchers to collect tissue samples to study cell biology including gene expression, histological analysis, signaling pathways, and protein translation. This article describes two techniques for biopsy of the bovine mammary gland (MG). Three healthy Holstein dairy cows were the subjects. Before biopsies, cows were milked and subsequently restrained in a cattle chute. An analgesic (flunixin meglumine, 1.1 to 2.2 mg/kg of body weight) was administered via jugular intravenous [IV] injection 15-20 min prior to biopsy. For standing sedation, xylazine hydrochloride (0.01-0.05 mg/kg of body weight) was injected via the coccygeal vessels 5-10 min before the procedure. Once adequately sedated, the biopsy site was aseptically prepared and locally anaesthetized with 6 mL of 2% lidocaine hydrochloride via subcutaneous injection. Using aseptic technique, a 2 to 3 cm vertical incision was made using a number 10 scalpel. Core and needle biopsy tools were used. The core biopsy tool was attached to a cordless drill and inserted into the MG tissue through the incision using a clock-wise drill action. The needle biopsy tool was manually inserted into the incision site. Immediately after the procedure, an assistant applied pressure on the incision site for 20 to 25 min using a sterile towel to achieve hemostasis. Stainless steel surgical staples were used to oppose the skin incision. The staples were removed 10 days post-procedure. The main advantages of core and needle biopsies is that both approaches are minimally invasive procedures that can be safely performed in healthy cows. Milk yield following the biopsy was unaffected. These procedures require a short recovery time and result in fewer risks of complications. Specific limitations may include bleeding after the biopsy and infection on the biopsy site. Applications of these techniques include tissue collection for clinical diagnosis and research purposes, such as primary cell culture.

This article presents a bovine mammary gland biopsy using core and needle biopsy tools. Harvested tissue can be used for cell culture or to assess mammary physiology and metabolism including gene expression, protein expression, protein modifications, immunohistochemistry, and metabolite concentrations.

소 유선 생 검 기술

Bovine mammary gland biopsies allow researchers to collect tissue samples to study cell biology including gene expression, histological analysis, ...

Longitudinal Intravital Microscopy Using a Mammary Imaging Window with Replaceable Lid

The branched structure of the mammary gland is highly dynamic and undergoes several phases of growth and remodeling after birth. Intravital microscopy in combination with skin flap surgery or implantation of imaging windows has been used to study the dynamics of the healthy mammary gland at different developmental stages. Most mammary imaging technologies are limited to a time frame of hours to days, whereas the majority of mammary gland remodeling processes occur in time frames of days to weeks. To study mammary gland remodeling, methods that allow optical access to the tissue of interest for extended time frames are required. Here, an improved version of the titanium mammary imaging window with a replaceable lid (R.MIW) is described that allows high-resolution imaging of the mammary gland with a cellular resolution for up to several weeks. Importantly, the R.MIW provides tissue access over the entire duration of the intravital imaging experiment and could therefore be used for local tissue manipulation, labeling, drug administration, or image-guided microdissection. Taken together, the R.MIW enables high-resolution characterization of the cellular dynamics during mammary gland development, homeostasis, and disease.

This protocol describes a novel mammary imaging window with a replaceable lid (R.MIW). Intravital microscopy after implantation of the R.MIW allows for longitudinal and multi-day imaging of the healthy and diseased mammary gland with a cellular resolution during the different developmental stages.

교체 가능한 뚜껑이있는 유방 이미징 창을 사용하는 종방향 생체 내 현미경 검사

The branched structure of the mammary gland is highly dynamic and undergoes several phases of growth and remodeling after birth. Intravital microscopy in ...