Çalışma tarafından desteklenen ABD Kamu Sağlığı Servisi Hibe DK-081.527, DK-072.473, Chicago Diyabet Araştırma ve Eğitim Merkezi (Hayvan modeller Core) Üniversitesi ve Kovler Aile Vakfı hediye DK-20.595.

Bölüm 1: Örneklerin Hazırlanması <ol><li> Diseksiyon mikroskobu altında bir fare koyun, hala bağlı duodenum ve dalak ile birlikte tüm pankreas kaldırmak ve önceden tartılmış bir cam slayt organları. Onikiparmak bağırsağı, pankreas bağlı olduğu kenarındaki bağ doku keserek çıkarın. Dalak ve pankreas aşırı yağ pankreas slayt tamamen izole kadar kesin ve çıkarın. (Pankreas yağ, pankreas dokusu ve biraz daha koyu ve bronzlaştırıcı bir renk ayırt beyaz renk ile ayırt edilebilir.) </li><li> Tamamen kapalı şekilde pankreas üzerine bir lamel (standart kapak cam 25x75mm 50x75mm geniş cam kapak) yerleştirin. Slayt ve kapağı arasındaki pankreas düzleştirmek için bir kilo (ağır bir kitap gibi) kullanın. Pankreas düzleşme, ağırlığına bağlı olarak yaklaşık beş dakika sürer. </li><li> Ağırlık çıkarınız. Pankreas slayt karşı iyi düzleştirilmiş ise% 4 paraformaldehid (PFA), 4 ° C gecede yerleştirin. Sabah lamelleri kaldırmak ve PFA için tüm pankreas gün (6-8 saat) sırasında maruz. Pankreas iyi sabit değilse, daha uzun süre kalır PFA muhtemelen gecede izin. % 1 ile konteyner Transfer slaytlar Triton fosfat x-100 (PBS) gece tamponlu salin. Ertesi gün, 1-2 gün için doymuş sakkaroz konteyner slaytlar transfer. Sonra floresan sinyallerin en uygun çözünürlüğü sağlar doku temizlenir kadar% 100 gliserol bir konteyner, slaytlar, doku temizleme yaklaşık 1-3 gün sürer. (Floresan sinyalleri hatta haftalar ve aylar sürebilir, ancak doku optimum çözünürlük görüntüleme temizlendikten sonra en kısa sürede yapılmalıdır.) </li><li> Karanlık ve serin bir alanda pankreas organ ve floresan sinyalleri korumak için saklayın. </li></ol> Bölüm 2: Görüntüleme ve sağlam pankreas beta hücre alanı niceleme <ol><li> Forseps veya eldivenli ellerini kullanarak,% 100 gliserol ile konteyner slayt çıkarmak. Aşırı gliserol silme ve sadece etanol ile arka kapak cam temizleme görüntüleme için slayt hazırlayın. </li><li> StereoInvestigator yazılımı (MicroBrightField, Williston, VT) açın ve görüntüleme fareler için 2x objektif lens seçmek, 3 haftadan daha büyük cam slaytlar büyük pancreata veya görüntüleme fareler için 10x objektif lens iki 25mm lamelleri arasında sıkışan 3 hafta daha genç pancreata. (Image → Canlı Görüntü) görüntü yaşamak için ayarlayarak görüntü gözünüzde canlandırın. GFP etiketli beta-hücrelerinin açıkça gösteren bir pozlama süresi seçin. (Görüntü overexposing ek yanlış sinyaller oluşturur görüntü Underexposing, küçük adacıklar ve beta-hücrelerinin hariç.) </li><li> Bir referans noktası oluşturmak ve tüm pankreasın etrafında bir kontur çizmek için devam etmek için ekrandaki herhangi bir yere tıklayın. Sanal Slice işlemi (Image → Sanal Dilim Edinme) başlatın. Belirlemek ve en iyi odak düzlemi, yani odak düğmesini kaydırarak Z ekseni üzerinde görünür ve odaklı adacıklar ve beta-hücrelerinin en fazla sayıda uçağın seçin. Seçildikten sonra, StereoInvestigator otomatik olarak ekranda görüntülenir görüntü yakalar. Motorlu bir sahne ile, Sanal Slice özelliği sırayla çizilmiş kontür (Şekil 1A) içerisinde ayrı bir görüntü olarak her optik panel yakalar ve sonra birleştirilmiş bir görüntü (Şekil 1B) olarak hepsini birleştirir. Sanal Dilim görüntüleme epifluorescent yapılandırmasını kullanır son Sanal Dilim maksimum entegre bir iki boyutlu bir görüntü o kadar kamera mükemmel bir odak değil, olanlar da dahil olmak üzere, belli bir derinlikte bulunan tüm sinyaller, yakalamanızı sağlar, yani widefield filtreleri tüm pankreas üç boyutlu rekonstrüksiyon projeksiyon. Ortalama Sanal Dilim tarama süresi örnek başına 30 dakika, ancak büyük bir pankreas için bir saat kadar sürebilir. </li><li> Görüntüyü kaydettikten sonra, ImageJ ile görüntü açarak analizi başlar. Görüntünün yüklenmesi tamamlandıktan sonra ekranda göründüğünde, açık ve Sanal Dilim analiz makro (çalıştırmak <a href="http://www.jove.com/files/ftp_upload/1970/Supplementary_material 1.pdf">Ek malzeme 1</a> ). </li><li> Makro tarafından verilen adım adım talimatları uygulayın. &quot;Sihirli Değnek&quot; ve &quot;Select Alanı&quot; araçları ile ışık yayılımı tarafından oluşturulan istenmeyen eserler ortadan kaldırın. Görüntü gereksiz arka plan kontrol edin ve çıkarın. Izleyen Rolling Balo özelliği etkin otofloresans ve dağınık ışık kaldırmak ve böylece &quot;Line&quot; aracı ile en büyük adacık yarıçapı belirleyin. Küçük adacık beta-hücrelerinin ve kümelerin floresan sinyallerini yakalamak için en uygun &quot;Düşük Eşik&quot; Test ve seçmek (&lt;3x10 4 mikron 2). Test ve adacıklar floresan sinyallerini yakalamak için en uygun &quot;Yüksek Eşik&quot; seçeneğini seçin. Sanal Dilim ölçecek miktarının, Çalıştırve adacık alan, çevre, dairesellik ve Feret çapı bir liste oluşturur. </li></ol><img src="/files/ftp_upload/1970/1970Fig1A.jpg" alt="Şekil 1a&#39;da" /> Şekil 1A sağlam bir yetişkin pankreas beta-hücrelerinin tüm dağıtım Sanal Dilim görüntü analizi 10 haftalık bir erkek fare pankreas bireysel optik paneller Görüntüler 2x amacı altına alınmış. Görüntüleri tüm dağıtım, adacık beta-hücrelerinin (&lt;10 hücre), hatta küçük kümeleri içerir. <img src="/files/ftp_upload/1970/1970Fig1B.jpg" alt="Şekil 1b" /> Şekil 1B sağlam bir yetişkin pankreas beta-hücrelerinin tüm dağıtım Sanal Dilim görüntü analizi Sağ dorsal pankreas ve sol ventral pankreas ile birleşik bir sanal dilim. Ölçek çubuğu 5 mm. Temsilcisi Sonuçlar Sanal Dilim görüntüleme için bir pankreas Yeterli hazırlık etkili bir ölçümü için izin GFP-açık ve seçik adacıklar Sanal Slice görüntü olarak bir bütün pankreas beta-hücrelerinin etiketli. In situ tüm pankreas Başarılı Sanal Dilim görüntüleme, sadece bireysel ve toplam adacık alan, ama aynı zamanda adacık boyut dağılımı, bölgesel analizi ve büyüme modelleri de beta-hücrelerinin, incelemek ve ölçmek için araçlar sağlar . ImageJ Sanal Dilim görüntüleri analiz tüm beta-hücrelerinin aynı anda tespit etme kapasitesine korur iken, aynı zamanda, belirli bir adacık boyutları (alan veya çapı) analiz kısıtlayan veya konuma göre ölçmek için beta hücreleri kontrol edebilirsiniz. Standart bir analiz eserler örneğin, tek bir beta-hücre (&lt;170 mikron 2) ve kayıtları alan, çevre (bir alanı çevreleyen mesafe), dairesellik (1.0 mükemmel bir daire temsil yuvarlaklık bir derece) daha küçük moloz, dışlar ve her adacık Feret çapı (bir alanda en uzun mesafe) tespit edildi. Sanal Dilim Havza segmentasyon, istihdam şekillerine göre bireysel adacıklar halinde üst üste adacıklar ayıran ve ayıran sanal bir dilim görüntüleri artırmak analiz yeteneğine sahiptir. Bu analiz, daha ayrıntılı görüntüler bir dizi üretir; bu alçak ve yüksek yoğunluklu (Şekil 1C) hem de her biri ayrı ayrı numaralandırılır ve ilgili bilgiler bir tablo listelenir (Şekil altında görüntü maskeleri, tüm varsayılan adacıklar bir taslak içerir 1D) ve orijinal Sanal Dilim görüntü (Şekil 1B). Bu potansiyel teknik önyargı çarpık karşılıksız ışık da dahil olmak üzere büyük yapıları çevreleyen adacıkları miktarının, adacıklar, küçük parçacıklar, özellikle zayıf sinyaller toplam sayısını azaltarak eşik değerleri seçerken dikkat edin. <img src="/files/ftp_upload/1970/1970Fig1C.jpg" alt="Şekil 1.C" /> Sağlam bir yetişkin pankreas beta-hücrelerinin tüm dağıtım Şekil 1C Sanal Dilim görüntü analizi Sanal dilim floresan parçacıkların bir maske. Renk kodları: [1] mavi: düşük yoğunluklu floresan; [2] yeşil: orta yoğunluğu floresan; ve [3] kırmızı: yüksek yoğunluklu floresan. <img src="/files/ftp_upload/1970/1970Fig1D.jpg" alt="Şekil 1.d" /> Resim sağlam bir yetişkin pankreas, beta-hücrelerinin tüm dağıtım 1D Virtual Dilim resim analizi Bireysel adacıklar / beta-hücrelerinin kümeleri Niceleme. Her bir adacık (beta hücreleri küçük kümeler dahil olmak üzere) karşılık gelen bir grafik ayrıntılı bilgi sayılı olduğunu unutmayın. (3) daireselliği, (2) çevre (1) alanı: her yapı için dört parametre alındı ​​yuvarlaklığını bir ölçüde sayısı 1.0 mükemmel bir daire temsil eder ve (4) Feret çapı: bir alan içinde en uzun mesafe. # 706 sadece bir kaç beta-hücrelerinin bir alana sahip analizleri çözünürlüklü ekranlara olduğunu unutmayın. Havza segmentasyon, bitişik adacık grupları gibi gösterildiği gibi algılar ve uygun ayrı adacıklar (adacıklar, 1554, 1555, ve 1556) gibi onları ayırır. <img src="/files/ftp_upload/1970/1970Fig1E.jpg" alt="Şekil 1e" /> Şekil 1E sağlam bir yetişkin pankreas beta-hücrelerinin tüm dağıtım Sanal Dilim görüntü analizi Alanı, çevre ve Feret çapı eksenleri ile 3 boyutlu sanal dilim analiz arsa. Her nokta bir adacık temsil eder.

<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>

Sağlam pankreas adacık beta-hücre kümeleri Sanal Dilim görüntüleri analiz adacıklar tüm dağıtım geniş çaplı bir görünüm sağlar. Gelişmeler ve zamanla toplam adacık dağıtım değişiklikleri inceledi ve mukayese edilebilir. Böyle bir örnek, pankreas adacık oluşumu (1) bizim analizde gösterilmiştir. Çeşitli zaman noktalarında (P1-P21) yenidoğan farelerin pancreata kapsamlı bir analiz adacıklar fisyon oluşturduğu olduğunu göstermiştir. Beta-hücre çoğalması, P1-P10 farelerde logno...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<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>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>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

in situ quantification of pancreatic beta-cell mass in mice

Sağlık ve hastalık belirli hücre popülasyonlarının değişiklikler İzleme biyomedikal araştırmanın önemli bir hedeftir. Izleme pankreatik beta hücre proliferasyonu ve adacık büyüme süreci, özellikle zor. Biz ImageJ (rsb.info.nih.gov / ij /) için yazılmış bir makro floresan etiketli beta-hücrelerinin transgenik farelerin sağlam pankreas beta-hücrelerinin dağıtım yakalamak için bir yöntem geliştirdiler. Tüm pankreas, pankreas diseksiyonu ve doku temizleme ardından sonra GFP etiketli beta-hücrelerinin incelenir, sanal bir dilim olarak yakalanır. Analiz miktarının toplam beta-hücre alanı, adacık sayısı ve boyutu dağılımı için her adacık beta-hücrelerinin küçük kümeleri için belirli parametreler ve yerleri referans içerir. Adacık tüm dağıtım üç boyutlu olarak çizilen, boyutu ve şekli her adacık dağıtım bilgileri bir bakışta genel beta-hücre alanındaki değişikliklerin niceliksel ve niteliksel karşılaştırma sağlar.

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

Sanal kesit görüntüleme için aşağıdaki protokol pankreas diseksiyonu sürecini özetliyor ve sonraki tüm pankreas tüm GFP etiketli beta-hücrelerinin kantifikasyon.

Farelerde, pankreatik beta-hücre Kütle yerinde Niceleme

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 Görüntüleme.  University of Chicago. Sanal kesit görüntüleme için aşağıdaki protokol pankreas diseksiyonu sürecini özetliyor ve sonraki tüm pankreas tüm GFP etiketli beta-hücrelerinin kantifikasyon.

Video: 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.

Diyabetik Fare Böbrek Kapsül içine, Pankreas Islets Nakli

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 ...

Biyoloji

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.

İyonlaştırıcı Radyasyon Cevabı γH2AX Foci Kantitasyonu

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.

Bilgisayar destekli Büyük ölçekli Görselleştirme ve Pankreas Adacık Kütle, Boyut Dağılımı ve Mimarlık Niceleme

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.

Proteinler Peptid immunoaffiniti Zenginleştirme kullanarak Niceleme Kütle Spektrometresi ile birleştiğinde

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.

Ekstansör digitorum longus kas ve kas Fonksiyonunun Değerlendirilmesi<em&gt; Ex vivo</em&gt; Ve tibialis anterior kas<em&gt; Yerinde</em&gt; Farelerde

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.

Floresan kullanarak mRNA Moleküllerin Görselleştirme ve Analiz<em&gt; Yerinde</emIn&gt; Hibridizasyon<em&gt; Saccharomyces cerevisiae</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.

Mutlak Protein Niceleme Kütle Spektrometresi İzleme Seçilen Tepki

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.

Kristalin Selüloz Birikimin Yüksek Çözünürlüklü Niceleme<em&gt; Arabidopsis</em&gt; Kökler Doku spesifik Hücre Duvarı Değişiklikler Monitör

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).

Gözlem ve telomer Niceleme ve Tekrarlayan Diziler kullanma Floresan<em&gt; In Situ</emPNA Sondalar&gt; ile hibridizasyon (FISH)<em&gt; Caenorhabditis elegans</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.