연구는에서 지원하는 미국 보건 서비스 그랜트 DK - 081527, DK - 072473 시카고 당뇨병 연구 및 교육 센터 (동물 모델 코어) 대학 및 Kovler 가족 재단에서 선물 DK - 20595.

1 부 : 표본의 준비 <ol><li> 해부 현미경 아래에 마우스를 놓고 여전히 첨부된 십이지장과 비장과 함께 전체 췌장을 제거하고 미리 무게 유리 슬라이드에 장기 장소. 그것은 췌장에 바인딩된 어디 측면을 따라 자사의 결합 조직을 절단하여 십이지장을 제거합니다. 췌장이 완전히 슬라이드에 고립된 때까지 췌장에있는 비장과 여분의 지방을 잘라 제거합니다. (췌장 지방은 췌장 조직의 약간 어두운 컬러와 태너에서 차별은 흰색 색상에 의해 구분됩니다.) </li><li> 그것이 완전히 적용되도록 췌장을 통해, coverslip을 (표준 커버 유리 25x75mm 50x75mm 대형 커버 유리) 놓습니다. 슬라이드와 커버 사이의 췌장을 버리고 무게 (무거운 책을 예)를 사용합니다. 췌장을 평평화, 무게에 따라하면 약 5 분이 걸립니다. </li><li> 무게를 벗어. 췌장은 슬라이드에 대해 잘 평평 경우 4 ° C 하룻밤에 4 % paraformaldehyde (PFA)에 놓으십시오. 아침에 coverslips을 제거하고 잘 날 (6~8시간) 동안 PFA로 전체 췌장을 폭로. 췌장이 잘 고정되지 않을 경우, 그것이 더 이상 PFA에 남아 아마도 하룻밤 수 있습니다. 1퍼센트로 컨테이너에 슬라이드를 전송 튼과 인산염의 X - 100 (PBS) 야간 호수 버퍼. 그 다음날, 1-2 일 동안 포화 자당의 컨테이너에 슬라이드를 전송합니다. 그렇다면 형광 신호의 최적 해상도를위한 수있는 조직이 해결이 될 때까지 100 % 글리세롤의 용기에 슬라이드를 전송, 조직 삭제는 약 1-3일 소요됩니다. (형광 신호도 주, 몇 달 동안 계속 수도 있지만 조직이 최적의 해상도 허가 후 이미징은 곧 수행되어야합니다.) </li><li> 오르간과 형광 신호를 보존하기 위해 어둡고 시원한 지역에서 췌장을 저장합니다. </li></ol> 2 부 : 이미징 및 손상 췌장에있는 베타 세포 영역을 quantifying <ol><li> 포셉 또는 장갑 낀 손을 사용하여 100 % 글리세롤로 컨테이너의 슬라이드를 꺼내. 초과 글리세롤을 닦아 주네와 독점적으로 에탄올과 커버 유리의 뒷면을 청소하여 이미징에 대한 슬라이드를 준비합니다. </li><li> StereoInvestigator 소프트웨어 (MicroBrightField, 윌리 스턴, VT)을 열고 이미징 생쥐에 대한 2X 객관적 렌즈를 선택 대형 유리 슬라이드 3 주 이상 이전 pancreata, 또는 이미지 마우스에 대한 10X 목표 렌즈 두 25mm coverslips 사이에 누를 삼주 미만 pancreata. 그것이 (이미지 → 라이브 이미지) 이미지를 살고 설정하여 이미지를 시각화. 명확하게 GFP - 태그 베타 세포를 보여주는 노출 시간을 선택합니다. (이미지를 overexposing하면 추가 잘못된 신호를 생성하는 동안 이미지를 Underexposing하면 작은 아일 레츠 및 베타 세포에서 제외됩니다.) </li><li> 기준점을 설정하고 전체 췌장 주위에 윤곽을 그리는 진행 화면에 아무 곳이나 클릭합니다. 가상 슬라이스 작업 (이미지 → 가상 슬라이스를 취득)를 시작합니다. 최고의 초점 평면 즉, 초점 노브를 스크롤하여 Z 축에 표시하고 집중 아일 레츠와 베타 세포의 가장 높은 숫자가있는 비행기를 확인하고 선택합니다. 일단 선택, StereoInvestigator는 자동으로 화면에 표시되는 이미지를 캡처합니다. 전동 무대로 가상 슬라이스 기능은 순차적으로 그려진 등고선 (그림 1A) 내에서 뚜렷한 이미지로 각 광 패널을 캡처하고 다음 하나 통합 이미지 (그림의 1B)로 그들을 결합합니다. 가상 슬라이스 영상은 epifluorescent 구성을 사용하는 최종 가상 슬라이스가 최대되지 않은 통합 2 차원 이미지 수 있도록 카메라가 아니라 완벽하게 초점을 포함하여 특정 깊이에서 현재 모든 신호를, 캡처 수 있습니다 즉, 위드 필터 전체 췌장의 3 차원 재건의 투영. 평균 가상 슬라이스 스캔 시간은 샘플 30 분이지만, 대형 췌장에 대한 시간까지 걸릴 수 있습니다. </li><li> 이미지를 저장 후, I​​mageJ와 이미지를 개방하여 분석을 시작합니다. 이미지가 로딩을 완료하고 화면에 나타나면, 열고 가상 슬라이스 분석 매크로 (실행 <a href="http://www.jove.com/files/ftp_upload/1970/Supplementary_material 1.pdf">보충 자료 1</a> ). </li><li> 매크로에서 제공하는 단계별 지침을 따르십시오. &quot;매직 원드&quot;과 &quot;선택 영역&quot;도구를 사용하여 빛의 산란에 의해 만들어진 원치 않는 아티팩트를 제거합니다. 이미지에서 불필요한 배경을 확인하고 뺍니다. 장인 롤링 볼 기능이 효과적으로 autofluorescence 및 흩어져 빛을 제거할 수 있도록 &quot;라인&quot;도구를 사용하여 가장 큰 아일릿의 반경을 결정합니다. 베타 세포의 작은 아일 레츠와 클러스터의 형광 신호를 캡처하는 가장 적절한 &quot;낮은 임계값을&quot;테스트를 선택합니다 (&lt;3x10 4 μm의 2). 아일 레츠의 형광 신호를 캡처하는 가장 적절한 &quot;고 한계&quot;를 테스트하고 선택합니다. 측정되는 가상 슬라이스의 부량을 실행그리고 아일릿 면적, 둘레, 순환성, 그리고 Feret의 직경의 목록을 생산하고 있습니다. </li></ol><img src="/files/ftp_upload/1970/1970Fig1A.jpg" alt="그림 1A" /> 손상 성인 췌장의 베타 세포의 전체 분포의 그림 1A 가상 슬라이스 이미지 분석 10 주에서 남성 마우스 췌장의 개별 광학 패널의 이미지 2X 목적에 따라 촬영. 이미지 아일 레츠의 전체 분포, 베타 세포 (&lt;10 셀)의도 작은 클러스터를 포함합니다. <img src="/files/ftp_upload/1970/1970Fig1B.jpg" alt="그림 1B" /> 손상 성인 췌장의 베타 세포의 전체 분포의 그림 1B 가상 슬라이스 이미지 분석 오른쪽에있는 등 지느러미 췌장과 왼쪽에있는 복부 췌장과 통일된 가상 조각. 스케일 바 5mm이다. 대표 결과 가상 슬라이스 이미지에 대한 췌장의 능숙 준비의 모든 효과에 대한 부량 수 GFP - 태그를 명확하고 뚜렷한 아일 레츠와 가상 슬라이스 이미지로 전체 췌장의 베타 세포를. 현장에있는 전체 췌장의 성공적인 가상 슬라이스 영상뿐만 아니라 개인과 전체 아일릿 지역뿐만 아니라 아일릿 크기 분포, 지역 분석, 성장 패턴뿐만 아니라 검사와 베타 세포를 계량하는 방법을 제공합니다. ImageJ는 가상 슬라이스 이미지의 분석에서 동시에 모든 베타 세포를 감지하는 능력을 유지하는 동안, 또한 특정 아일릿 크기 (면적 또는 직경에 의해)로 분석을 제한, 또는 위치에 의해 계량하는 베타 세포를 제어할 수 있습니다. 표준 분석 유물 예를 들어, 하나의 베타 세포 (&lt;170 μm의 2) 및 기록 영역, 경계 (주변 거리), 순환성 (1.0 완벽한 원형을 나타냅니다 진원도의 정도)보다 작은 파편을 제외 각 아일릿에 대한 Feret의 직경 (지역 가장 긴 거리) 발견했습니다. 가상 슬라이스 분석들은 모양에 따라 개별 아일 레츠에 중복 아일 레츠를 구분하고 구분 워터셰트 세그먼트를 채용하여 가상 슬라이스 이미지를 향상시킬 수 있습니다. 분석 더욱 자세한 이미지의 일련의 생산, 이것은 각각 개별적으로 번호와 해당 정보 테이블에 나열된 (그림 아르 낮은 높은 강도 (그림 1C) 모두에서 이미지의 마스크, 모든 추정 아일 레츠의 개요를 포함 . 1D), 그리고 원래 가상 슬라이스 이미지 (그림의 1B). 스큐 큰 구조를 둘러싼 무료의 빛을 포함하여 아일 레츠의 부량하거나, 아일 레츠, 작은 입자 특히 희미한 신호의 총 수를 줄임으로써 임계 값을 선택하면 그 잠재적인 기술 편견을합니다. <img src="/files/ftp_upload/1970/1970Fig1C.jpg" alt="그림 1C" /> 손상 성인 췌장의 베타 세포의 전체 분포의 그림 1C 가상 슬라이스 이미지 분석 가상 슬라이스의 형광 입자의 마스크. 색상 코드 : [1] 파란색 : 낮은 강도의 형광, [2] 녹색 : 중간 강도의 형광 및 [3] 빨간 : 높은 강도의 형광. <img src="/files/ftp_upload/1970/1970Fig1D.jpg" alt="그림 1D" /> 손상 성인 췌장의 베타 세포의 전체 분포의 1D 가상 슬라이스 이미지 분석을 그림 개인 아일 레츠 / 베타 - 세포 클러스터의 부량. 각 아일릿이 (베타 - 세포의 작은 클러스터를 포함) 해당 차트에 대한 자세한 정보는, 번호입니다. (3) 순환성 (2) 경계 (1) 면적 : 네 개의 매개 변수는 각 구조에 찍은 숫자 1.0는 완벽한 원형을 나타냅니다 진원도의 정도, 그리고 (4) Feret의 직경 : 지역 가장 긴 거리. # 706은 몇 베타 세포의 면적 분석 해상도를 표시합니다. 워터셰트 세분화는 그림과 같이 인접 아일 레츠 그룹을 감지하고 적절하게 독특한 아일 레츠 (아일 레츠 1554, 1555 및 1556)로 그들을 구별. <img src="/files/ftp_upload/1970/1970Fig1E.jpg" alt="그림 1E" /> 손상 성인 췌장의 베타 세포의 전체 분포의 그림 1E 가상 슬라이스 이미지 분석 면적, 둘레, 그리고 Feret의 직경 축과 가상 슬라이스 분석 3 차원 플롯. 각 점은 아일릿을 나타냅니다.

<ol>
	<li>Miller, K., Kim, A., Kilimnik, G., Moka, U., Jo, J., Periwal, V., Hara, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Islet+formation+during+the+neonatal+development+in+mice.">Islet formation during the neonatal development in mice.</a> PloS One. 4, E7739-E7739 (2009).</li><li>Kilimnik, G., Kim, A., Jo, J., Miller, K., Hara, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+pancreatic+islet+distribution+in+situ+in+mice.">Quantification of pancreatic islet distribution in situ in mice.</a> Am J Physiol Endocrinol Metab. 297, E1331-E1338 (2009).</li><li>Hara, M., Wang, X., Kawamura, T., Bindokas, V. P., Dizon, R. F., Alcoser, S. Y., Magnuson, M. A., Bell, G. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+mice+with+green+fluorescent+protein-labeled+pancreatic+beta+-cells.">Transgenic mice with green fluorescent protein-labeled pancreatic beta -cells.</a> Am J Physiol Endocrinol Metab. 284, E177-E183 (2003).</li><li>Hara, M., Dizon, R. F., Glick, B. S., Lee, C. S., Kaestner, K. H., Piston, D. W., Bindokas, V. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+pancreatic+beta-cells+in+the+intact+pancreas.">Imaging pancreatic beta-cells in the intact pancreas.</a> Am J Physiol Endocrinol Metab. 290, E1041-E1047 (2006).</li></ol>

관심 없음 충돌 선언하지 않습니다.

손상 췌장에서 아일 레츠와 베타 세포 클러스터의 가상 슬라이스 이미지의 분석 아일 레츠의 전체 분포의 대규모보기를 제공합니다. 개발 및 시간이 지남에 따라 전체 아일릿 분포의 변화에​​ 따라서 공부하고 비교할 수 있습니다. 이러한 예제는 췌장 아일릿 형성 (1) 우리의 분석에 증명됩니다. 여러 시간 지점 (P1 - P21)에서 신생아 쥐 pancreata의 포괄적인 분석 아일 레츠는 분열에 의해 형성되는...

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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Forceps</td>
<td>&nbsp;</td>
<td>Miltex</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Scissors</td>
<td>&nbsp;</td>
<td>F.S.T</td>
<td>14001-13</td>
<td>01n2</td>
</tr>
<tr>
<td>Scissors</td>
<td>&nbsp;</td>
<td>F.S.T</td>
<td>14072-10</td>
<td>02s5	</td>
</tr>
<tr>
<td>Large glass slide</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>71862-01</td>
<td>75x51x1.2mm</td>
</tr>
<tr>
<td>Large cover glass</td>
<td>&nbsp;</td>
<td>Ted Pella, Inc</td>
<td>260462</td>
<td>50x75mm</td>
</tr>
<tr>
<td>Glass Slide</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>12-550-15</td>
<td>25x75x1.0mm</td>
</tr>
<tr>
<td>Cover glass</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>12-458-5M</td>
<td>75x25mm</td>
</tr>
<tr>
<td>Cover glass</td>
<td>&nbsp;</td>
<td>Erie Scientific</td>
<td>&nbsp;</td>
<td>25mm circle</td>
</tr>
<tr>
<td>Fluorescent microscope</td>
<td>&nbsp;</td>
<td>Olympus IX-81</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Dissection microscope</td>
<td>&nbsp;</td>
<td>Olympus SZX12</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Stereo Investigator</td>
<td>&nbsp;</td>
<td>MicroBrightField</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>MIP-GFP mice</td>
<td>&nbsp;</td>
<td>Jackson Laboratory</td>
<td>&nbsp;</td>
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in situ quantification of pancreatic beta-cell mass in mice

건강과 질병의 특정 세포 인구의 변화를 추적하는 것은 생명 의학 연구의 중요한 목표입니다. 모니터링 췌장 베타 세포 증식과 아일릿 성장의 과정은 특히 도전이다. 우리는 ImageJ (rsb.info.nih.gov / 엘제이 /)에 대해 작성된 매크로와 형광 - 태그 베타 세포와 유전자 변형 생쥐의 췌장 손상에서 베타 세포의 분포를 캡처하는 방법을 개발했습니다. 췌장 해부 및 조직 삭제에 따라, 전체 췌장은 어떤 후 GFP - 태그 베타 세포 검사 있으며, 가상의 슬라이스로 캡처합니다. 분석 총 베타 세포 지역 아일릿 번호 및 각 아일릿과 베타 세포의 작은 클러스터에 대한 특정 매개 변수와 위치에 대한 참조와 크기 분포의 부량을 포함합니다. 아일 레츠의 전체 분포는 3 차원에 역모를, 각 아일릿의 크기와 모양에 대한 배포의 정보를 한 눈에 전체 베타 세포 분야에서 변화의 양적 및 질적 비교를 허용하실 수 있습니다.

Watch this Scientific Journal Video about In situ Quantification of Pancreatic Beta-cell Mass in Mice at JoVE.com

In situ Quantification of Pancreatic Beta-cell Mass in Mice

다음 프로토콜은 가상 슬라이스 이미지에 대한 췌장 해부 과정을 설명하고, 전체 췌장에있는 모든 GFP - 태그 베타 세포의 후속 부량.

마우스의 췌장 베타 세포 질량의 원위치 부량에서

Tracing changes of specific cell populations in health and disease is an important goal of biomedical research. The process of monitoring pancreatic ...

in-situ-quantification-of-pancreatic-beta-cell-mass-in-mice

Research

JoVE Journal

Biology

In situ Quantification of Pancreatic Beta-cell Mass in Mice

12.1K 조회수.  University of Chicago. 다음 프로토콜은 가상 슬라이스 이미지에 대한 췌장 해부 과정을 설명하고, 전체 췌장에있는 모든 GFP - 태그 베타 세포의 후속 부량.

비디오: In situ Quantification of Pancreatic Beta-cell Mass in Mice

Transplantation of Pancreatic Islets Into the Kidney Capsule of Diabetic Mice

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of diabetic or normal mice. We found that it was easier to concentrate the islets or cells into pellets in the final delivery tubing (PE50) used to transplant the cells under the kidney capsule. This technique provides both speed and ease while reducing any undue stress to the cells or to the mouse.

Loading: Settled, hand picked, islets or pelleted cells are carefully aspirated off the bottom of a 1.5 mL microcentrifuge tube using a p200 pipetteman and a straight, thin-wall pipette tip. A length of PE50 tubing is attached to the pipette tip using a small silicone adapter tubing. Cells are allowed to settle, in the tip, and then are transferred to the PE50 tubing by slowly dialing the pipetteman. Once the cells are near the end of the PE50 tubing, a kink is made and the silicone adaptor tubing is placed over the kink. The PE50 tubing is transferred to a 15 mL conical containing a cut 5 mL pipet, and the PE50 tubing is taped over the side of the 5 mL pipet to prevent curling during centrifuging. Cells are allowed to reach 1,000 rpm and stopped.
Transplantation: Recipient mice are anesthetized, shaved, and cleaned. A small incision is made on the left flank of the mouse and the kidney is exposed. The kidney, fat, and tissue are kept moist with normal saline swab. The distal end of the PE50 is attached to a Hamilton screw drive syringe, containing a pipette tip, using the silicone adaptor tubing. A small nick is made on the right flank side of the kidney, not too large nor too deep. The beveled end of the PE50 tubing, nearest the cells, is carefully placed under the capsule, the tubing is moved around gently to make space while swabbing normal saline; a dry capsule can tear easily. A small air bubble is delivered under the capsule by slowly dialing the syringe screw drive. Islets are then slowly delivered behind the air bubble. Once the islets have been delivered kidney homeostasis is maintained and the knick is cauterized with low heat. The kidney is placed back into the cavity and the peritoneum and skin are sutured and stapled. Mice are immediately treated with Flunixin and Buprenorphine s.q. and placed in a cage on a heating pad.

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of mice. Cells are concentrated into pellets in the final tubing used for transplanting the cells under the kidney capsule. The ease of this technique reduces stress to the cells and the mouse.

당뇨병 마우스의 신장 캡슐으로 췌장 아일 레츠의 이식

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of diabetic or normal mice.  We found that it was easier ...

연구

생물학

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &amp;#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

Ionising 방사선에 대한 응답으로 γH2AX Foci의 부량

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Computer-assisted Large-scale Visualization and Quantification of Pancreatic Islet Mass, Size Distribution and Architecture

The pancreatic islet is a unique micro-organ composed of several hormone secreting endocrine cells such as beta-cells (insulin), alpha-cells (glucagon), and delta-cells (somatostatin) that are embedded in the exocrine tissues and comprise 1-2% of the entire pancreas. There is a close correlation between body and pancreas weight. Total beta-cell mass also increases proportionately to compensate for the demand for insulin in the body. What escapes this proportionate expansion is the size distribution of islets. Large animals such as humans share similar islet size distributions with mice, suggesting that this micro-organ has a certain size limit to be functional. The inability of large animal pancreata to generate proportionately larger islets is compensated for by an increase in the number of islets and by an increase in the proportion of larger islets in their overall islet size distribution. Furthermore, islets exhibit a striking plasticity in cellular composition and architecture among different species and also within the same species under various pathophysiological conditions. In the present study, we describe novel approaches for the analysis of biological image data in order to facilitate the automation of analytic processes, which allow for the analysis of large and heterogeneous data collections in the study of such dynamic biological processes and complex structures. Such studies have been hampered due to technical difficulties of unbiased sampling and generating large-scale data sets to precisely capture the complexity of biological processes of islet biology. Here we show methods to collect unbiased "representative" data within the limited availability of samples (or to minimize the sample collection) and the standard experimental settings, and to precisely analyze the complex three-dimensional structure of the islet. Computer-assisted automation allows for the collection and analysis of large-scale data sets and also assures unbiased interpretation of the data. Furthermore, the precise quantification of islet size distribution and spatial coordinates (i.e. X, Y, Z-positions) not only leads to an accurate visualization of pancreatic islet structure and composition, but also allows us to identify patterns during development and adaptation to altering conditions through mathematical modeling. The methods developed in this study are applicable to studies of many other systems and organisms as well.

Novel computer-assisted methods of large-scale procurement and analysis of immunohistochemically stained pancreatic specimens are described: (1) Virtual Slice capture of the entire section; (2) Mass analysis of large-scale data; (3) Reconstruction of 2D Virtual Slices; (4) 3D islet mapping; and (5) Mathematical analysis.

컴퓨터를 이용한 대규모 시각화 및 췌장 아일릿 질량, 크기 유통과 건축의 부량

The pancreatic islet is a unique micro-organ composed of several hormone secreting endocrine cells such as beta-cells (insulin), alpha-cells (glucagon), ...

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, 'hook-effect').1 An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).2 In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules 3, 4 and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.5-7 To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.8-13 In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.

Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution mass spectrometry (MRM-MS) to provide quantitative measurement of peptides as surrogates for their respective proteins. Here we describe the protocol using magnetic particles in a partially automated format.

펩타이드 면역 친 화성 농축을 사용하여 단백질의 양을 정함은 질량 분광법과 함께

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in ...

Evaluation of Muscle Function of the Extensor Digitorum Longus Muscle Ex vivo and Tibialis Anterior Muscle In situ in Mice

Body movements are mainly provided by mechanical function of skeletal muscle. Skeletal muscle is composed of numerous bundles of myofibers that are sheathed by intramuscular connective tissues. Each myofiber contains many myofibrils that run longitudinally along the length of the myofiber. Myofibrils are the contractile apparatus of muscle and they are composed of repeated contractile units known as sarcomeres. A sarcomere unit contains actin and myosin filaments that are spaced by the Z discs and titin protein. Mechanical function of skeletal muscle is defined by the contractile and passive properties of muscle. The contractile properties are used to characterize the amount of force generated during muscle contraction, time of force generation and time of muscle relaxation. Any factor that affects muscle contraction (such as interaction between actin and myosin filaments, homeostasis of calcium, ATP/ADP ratio, etc.) influences the contractile properties. The passive properties refer to the elastic and viscous properties (stiffness and viscosity) of the muscle in the absence of contraction. These properties are determined by the extracellular and the intracellular structural components (such as titin) and connective tissues (mainly collagen) 1-2. The contractile and passive properties are two inseparable aspects of muscle function. For example, elbow flexion is accomplished by contraction of muscles in the anterior compartment of the upper arm and passive stretch of muscles in the posterior compartment of the upper arm. To truly understand muscle function, both contractile and passive properties should be studied.
The contractile and/or passive mechanical properties of muscle are often compromised in muscle diseases. A good example is Duchenne muscular dystrophy (DMD), a severe muscle wasting disease caused by dystrophin deficiency 3. Dystrophin is a cytoskeletal protein that stabilizes the muscle cell membrane (sarcolemma) during muscle contraction 4. In the absence of dystrophin, the sarcolemma is damaged by the shearing force generated during force transmission. This membrane tearing initiates a chain reaction which leads to muscle cell death and loss of contractile machinery. As a consequence, muscle force is reduced and dead myofibers are replaced by fibrotic tissues 5. This later change increases muscle stiffness 6. Accurate measurement of these changes provides important guide to evaluate disease progression and to determine therapeutic efficacy of novel gene/cell/pharmacological interventions. Here, we present two methods to evaluate both contractile and passive mechanical properties of the extensor digitorum longus (EDL) muscle and the contractile properties of the tibialis anterior (TA) muscle.

Evaluation of Muscle Function of the Extensor Digitorum Longus Muscle Ex vivo and Tibialis Anterior Muscle In situ in Mice

Changes in limb muscle contractile and passive mechanical properties are important biomarkers for muscle diseases. This manuscript describes physiological assays to measure these properties in the murine extensor digitorum longus and tibialis anterior muscles.

신근 Digitorum의 Longus 근육의 근육 기능의 평가<em&gt; 예 생체</em&gt;와 Tibialis 앞쪽에 근육<em&gt; 현장에서</em&gt; 마우스에

Body  movements are mainly provided by mechanical function of skeletal muscle. Skeletal  muscle is composed of numerous bundles of myofibers that are ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

형광을 사용하여 mRNA의 분자의 시각화 및 분석<em&gt; 제자리에서</em에서&gt; 하이브리드<em&gt; 사카 cerevisiae의</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This protocol provides step-by-step instructions for the development and application of quantitative assays using selected reaction monitoring (SRM) mass spectrometry (MS). First, likely quantotypic target peptides are identified based on numerous criteria. This includes identifying proteotypic peptides, avoiding sites of posttranslational modification, and analyzing the uniqueness of the target peptide to the target protein. Next, crude external peptide standards are synthesized and used to develop SRM assays, and the resulting assays are used to perform qualitative analyses of the biological samples. Finally, purified, quantified, heavy isotope labeled internal peptide standards are prepared and used to perform isotope dilution series SRM assays. Analysis of all of the resulting MS data is presented. This protocol was used to accurately assay the absolute abundance of proteins of the chemotaxis signaling pathway within RAW 264.7 cells (a mouse monocyte/macrophage cell line). The quantification of Gi2 (a heterotrimeric G-protein &#945;-subunit) is described in detail.

This protocol describes how to perform absolute quantification assays of target proteins within complex biological samples using selected reaction monitoring. It was used to accurately quantify proteins of the mouse macrophage chemotaxis signaling pathway. Target peptide selection, assay development, and qualitative and quantitative assays are described in detail.

절대 단백질 정량 질량 분석을 모니터링 선정 반응

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This ...

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix containing polysaccharides that include cellulose microfibrils that form both crystalline structures and cellulose chains of amorphous organization. The orientation of the cellulose fibers and their concentrations dictate the mechanical properties of the cell. Several methods are used to determine the levels of crystalline cellulose, each bringing both advantages and limitations. Some can distinguish the proportion of crystalline regions within the total cellulose. However, they are limited to whole-organ analyses that are deficient in spatiotemporal information. Others relying on live imaging, are limited by the use of imprecise dyes. Here, we report a sensitive polarized light-based system for specific quantification of relative light retardance, representing crystalline cellulose accumulation in cross sections of Arabidopsis thaliana roots. In this method, the cellular resolution and anatomical data are maintained, enabling direct comparisons between the different tissues composing the growing root. This approach opens a new analytical dimension, shedding light on the link between cell wall composition, cellular behavior and whole-organ growth.

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Crystalline cellulose is an important constituent of the plant cell wall. However, its quantification at a cellular resolution is technically challenging. Here, we report the use of polarized light technology and root cross sections to obtain information of cell wall composition at a spatiotemporal resolution.

에서 결정 셀룰로오스 축적의 높은 해상도 정량<em&gt; 애기 장대</em&gt; 뿌리 조직 특이 세포 벽 수정을 모니터링하는

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix ...

Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization (FISH) with PNA Probes in Caenorhabditis elegans

Telomere is a ribonucleoprotein structure that protects chromosomal ends from aberrant fusion and degradation. Telomere length is maintained by telomerase or an alternative pathway, known as alternative lengthening of telomeres (ALT)1. Recently, C. elegans has emerged as a multicellular model organism for the study of telomere and ALT2. Visualization of repetitive sequences in the genome is critical in understanding the biology of telomeres. While telomere length can be measured by telomere restriction fragment assay or quantitative PCR, these methods only provide the averaged telomere length. On the contrary, fluorescence in situ hybridization (FISH) can provide the information of the individual telomeres in cells. Here, we provide protocols and representative results of the method to determine telomere length of C. elegans by fluorescent in situ hybridization. This method provides a simple, but powerful, in situ procedure that does not cause noticeable damage to morphology. By using fluorescently labeled peptide nucleic acid (PNA) and digoxigenin-dUTP-labeled probe, we were able to visualize two different repetitive sequences: telomere repeats and template of ALT (TALT) in C. elegans embryos and gonads.

Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization FISH with PNA Probes in Caenorhabditis elegans

We report a concise procedure of fluorescence in situ hybridization (FISH) in the gonad and embryos of Caenorhabditis elegans for observing and quantifying repetitive sequences. We successfully observed and quantified two different repetitive sequences, telomere repeats and template of alternative lengthening of telomeres (TALT).

관측 및 텔로미어의 정량 및 반복 시퀀스 사용하여 형광<em&gt; 현장에서</emPNA 프로브에와&gt; 하이브리드 (FISH)<em&gt; 예쁜 꼬마 선충</em

Telomere is a ribonucleoprotein structure that protects chromosomal ends from aberrant fusion and degradation. Telomere length is maintained by telomerase ...

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to hyperglycemia and severe, life-threatening consequences. Understanding how the beta-cells operate under physiological conditions and what genetic and environmental factors might cause their dysfunction could lead to better treatment options for diabetic patients. The ability to measure calcium levels in beta-cells serves as an important indicator of beta-cell function, as the influx of calcium ions triggers insulin release. Here we describe a protocol for monitoring the glucose-stimulated calcium influx in zebrafish beta-cells by using GCaMP6s, a genetically encoded sensor of calcium. The method allows monitoring the intracellular calcium dynamics with single-cell resolution in ex vivo mounted islets. The glucose-responsiveness of beta-cells within the same islet can be captured simultaneously under different glucose concentrations, which suggests the presence of functional heterogeneity among zebrafish beta-cells. Furthermore, the technique provides high temporal and spatial resolution, which reveals the oscillatory nature of the calcium influx upon glucose stimulation. Our approach opens the doors to use the zebrafish as a model to investigate the contribution of genetic and environmental factors to beta-cell function and dysfunction.

Beta-cell functionality is important for blood-glucose homeostasis, which is evaluated at single-cell resolution using a genetically encoded reporter for calcium influx.