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

1.. Numune <ol><li> Üç gün laktasyon başlangıcı sonrası yabani tip CDH1 ikinci cerrahi meme bezleri gibi Igfbp7 null fareler tüketim. </li><li> Yerli meme bezi morfolojisi yeniden yardımcı cam slaytlar üzerine bezleri yayıldı. </li></ol> 2.. Fiksasyon ve Doku İşleme <ol><li> 4 ° C&#39;de o nötr tamponlu% 4 PFA O / N meme bezleri saptamak </li><li> Önce doku işleme% 70 etanol içinde bezleri saklayın. </li><li> Küçük doku işleme kaset bezleri aktarın. </li><li> Otomatik bir doku işlemci kullanarak dokular işleyin <ol><li> 45 dakika için ksilen içinde, 1 saat ve 2 kez% 100 etanol içinde, 45 dakika boyunca% 95 etanol içinde 45 dakika, 2 kez 3 kez% 70 etanol, etanol ve ksilen banyoları artan doku kurutmak. </li><li> Uygulanan basınç bir vakum içinde 1 saat boyunca her bir parafin ile 3 kez dokulara nüfuz. </li></ol></li><li> Blokları meydana getirmek için dokular parafin içinde Embed, Kesit için. </li></ol> 3.. Histoloji ve Blockface Görüntüleme <ol><li> Fazla parafin kaldırılana kadar bir döner mikrotom kullanılarak parafin blokları kesin. </li><li> Kasetine dik olan parafin bloğun en az iki köşe 1 mm delik için dikey bir freze makinesi kullanın. </li><li> Döner mikrotom üzerinde doku bloğu monte edin. </li><li> Mikrotom önündeki blockface görüntüleme sistemi 19 kurun. </li><li> Önce kesit optik blockface görüntü yakalayın. </li><li> Mikrotomu 5 mikron kalınlığında dört bölümden şeritler kesin. <ol><li> Soğuk su banyosuna şeritler aktarın. </li><li> Şerit ikinci ve dördüncü bölümleri ayırın ve mikroskop lamı üzerine takar. Ikinci ve dördüncü bölümleri seçimi bölümleri arasında bir 5 mm bir boşluk sağlar. </li><li> Bu mikroskop lamı üzerine yeniden monte sonra, bunu unwrinkle için sıcak su banyosunda (48 o C) her bölümü genişletin. NOT: CKesm, montaj bölümleri gibi gözyaşı, kat, çekme ve genişleme olarak bölümünde bazı bozulmalara neden unwrinkling. Bu eserler histoloji bölümlerin kayıt zorlaştırıyor. </li><li> Otomatik Stainer kullanılarak H &amp; E ile bölümleri Leke. </li><li> Otomatik coverslipper kullanarak slaytlar Coverslip. </li><li> Ilgi çözünürlükte dijital histoloji slayt tarayıcı kullanarak slaytlar sayısallaştır. Bu protokol için 20x büyütme ve çözünürlük 0.47 mm. </li><li> Blockface görüntüler, 18 mikron çözünürlüğü histoloji görüntü aşağı-örnek. </li></ol></li></ol> 4. Görüntü Kayıt <ol><li> Görüntü Segmentasyon ve nokta Seçimi <ol><li> Blockface görüntüler kayıt deliklerin piksel değerlerini ölçmek ve segmentinde parafin blok köşelerinde kayıt delikler sabit bir eşik olarak ortalama değeri kullanın. </li><li> Bazı ek parçalar da bölünmüş olabilir yanasabit eşik kullanarak, daireselliğe ve delikleri bulmak ve ekstra nesneleri atmak için segmente nesnelerin alanını kullanın. Bunu yapmak küçük bir kod yazmak ve bölümlere ayrılmış nesneler için (4π x alan) / (çevre) 2 oranını bulmak için. Yuvarlak nesneler için bu oranı 1&#39;dir. </li><li> Her meme bezi için, referans gibi bir blockface görüntüyü seçin ve kayıt delikler ve bölge-tabanlı kayıt teknikleri merkezi kullanarak referans blockface görüntülerin kalanını hizalayın. </li><li> Hizalanmış blockface görüntüler için, elle kesimi veya arka plan doku ayıklayın. Protokol kalanı için maske en büyükçe nesnesini kullanın. </li><li> H &amp; E bölümler için otomatik segmentasyon için aşağıdaki adımları uygulayın. <ol><li> Arka plan bölüm görüntüleri Otsu eşikleme tekniği 20 kullanın ve histoloji görüntülerin ikili maskeler oluşturabilirsiniz. </li><li> Belirlenmesi ve h kullanarak her maskesi içinde en büyük hacimli nesneyi seçinetiketli nesnelerin istogram. </li><li> Histoloji ve blockface maskeleri hem bir piksel genişliğinde sınır noktaları ayıklayın. </li><li> Parçalı doğrusal bir dizisi ile sınır noktaları temsil etmek, Zincir kodu algoritması 21 kullanın uyuyor. </li></ol></li></ol></li><li> İlk Sert Kayıt <ol><li> Ilk sert histoloji sınır noktaları ve bunlara karşılık gelen blockface görüntüleri arasında dönüştürmek bulmak için, Fourier tanımlayıcıları algoritma 22 kullanın. Bu ilk dönüştürmek çevirme, döndürme ve ölçek faktörleri içerir. </li><li> Başlangıç ​​önceki aşamada elde edilen dönüşüm her histoloji görseli dönüşümü. </li></ol></li><li> Sert Kayıt arıtma <ol><li> Yuvarlanan bir top filtre 23 kullanılarak histoloji konturundan yüksek eğrilik kenar bölümleri kaldırın. </li><li> Rastgele düzgün dağılım kullanılarak, kalan histoloji sınır noktalarından 500 puan seçin. </li><li> TransformFourier tanımlayıcıları elde edilen ilk dönüşüm ile histoloji rastgele sınır noktaları. </li><li> Blockface sınır noktaları bütün seti seçin ve blockface sınır noktaları, hedef ve histoloji rastgele sınır noktaları arasındaki sert dönüşüm bulmak için iteratif en yakın Puanlar (ICP) algoritması 24 kullanın. </li><li> Hizalanmış histoloji önceki adımda elde edilen görüntüleri ve hizalanmış histoloji görüntülerin yığını histoloji ses oluşturur Transform. </li><li> Histoloji hacminin görsel bir görüntü oluşturmak için bir 3D görselleştirme yazılımını kullanın. </li></ol></li><li> 5x büyütme Görüntüler Stack görüntüleniyor <ol><li> Büyütme 5x orijinal histoloji görüntü aşağı-örnek. </li><li> Histoloji görüntülerin birinde, ilgili bölgeyi Crop. </li><li> Kayıt iki aşamadan gelen katı dönüşümlerin kombinasyonunu kullanan diğer 5x histoloji görüntülerde bu bölgenin konumunu hesaplar. </li><li> Regi kırpınDiğer tüm histoloji görüntülerde aynı boyutta bölgeye ilgi ons. </li><li> Nihayet elle bölgeler arasındaki uyum rafine. Iki görüntüyü üst üste bindirir ve rotasyon ve diğer birinin üzerine görüntülerden birine çeviri için değerlerini seçmek için izin verir ve ardından hizalama kabul edilir dönüştürülmüş görüntüsünü kaydeden bir program yazın. </li><li> 3D görselleştirme yazılımı ile uyumlu 5x histoloji bölgelerin yığınları görüntüleyebilirsiniz. </li></ol></li></ol>

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The authors have nothing to disclose

Bu çalışmada, doku deforme olabilir dokusu içinde iç rastgele seçilmiş işaretlerini veya implante fiducial işaretleri, gerektirmeyen seri 2B histoloji görüntüleri, bir 3D histoloji cildini yeniden bir görüntü kaydı iş akışını geliştirdik. Tarif edilen yönteme göre, optik blockface görsel kendilerini önce kesit için, referans resim olarak kullanılır. Biz blockface görüntüleri hizalama yardımcı olmak üzere ve kameranın önüne parafin bloğun 2B çapraz hareketi için gidermesi parafin ...

IGFBP7 (7 bağlayıcı protein insülin benzeri büyüme faktörü), IGF-bağlayıcı protein ailesinin bir üyesidir ve IGF1 4 reseptörüne bağlandığı gösterilmiştir. Ksenograft tümör modellerinde IGFBP7 arasında büyük ölçüde yeniden yerleştirilmesi apoptoz ve hücre yaşlanması 7 endüksiyonu yoluyla 6 büyüme tümör inhibe ederken IGFBP7 arasında aşağı-düzenleme, meme kanseri 5 kötü prognoz ile ilişkili olduğu bilinmektedir. IGFPB7 etkilerini incelemek amacıyla, bir Igfbp7 null fare 5 (yayınlanmamış veri) oluşturuldu. Bu fareler tümörler gelişir yok ederken, onlar yumurtalık, kas ve karaciğer histolojik değişiklikleri yanı sıra meme bezi gelişim desenleme (yayınlanmamış veri) kusurları gösterir. Null fareler daha küçük çöp boyutları vardır ve birden fazla büyük ibrelerin (yayınlanmamış veri) sürdürmek edemiyoruz gibi kusurlu fenotip ilk belirtilmiştir. 3D histoloji hacimleri yararlı informat sağlamak potansiyeline sahiphacimsel Tıbbi görüntülerde patolojik bulguların sayısal ve karşılaştırmalı analizler ve değerlendirme için iyon. Üç boyutlu konfokal, iki-foton mikroskopi yerel ölçüde 14 de bezinin yüksek çözünürlüklü hücre morfolojik bilgi sağlayabilir, ancak bu bakış ve derinlik sınırlı bir alanı vardır. Histoloji hacim rekonstrüksiyonu çok daha büyük bir mekansal ölçüde üzerinde daha fazla bilgi sağlar. Bazı bozulması, büzülme, genişleme, gözyaşı ve kıvrımlar gibi histolojik kesitler, hazırlanması sırasında öngörülmektedir geleneksel yaklaşımları kullanma. Bu bozulmalar zor bir 3D hacmini yeniden bir 3D yığını içine seri histolojik görüntüleri kayıt olun. Defektli ardışık bölümlerin sayısı sağlam bölümler arasındaki benzerlikleri arttıkça azalır ve sonuç olarak kayıt işlemi daha karmaşık hale getirir. Farklı yöntemler histolojik kesitler kayıt ve sürekli bir histoloji vo yaratmak için ileri sürülmüştürlume. Bazı teknikler yoğunluk değişmeleri 8 bağlıdır ve diğer bölümleri 9 şekline dayanmaktadır. Bazı örnekler için anatomik yapılar dönüm tabanlı kayıt yöntemleri 12,13 ile birlikte görülecek 10,11 olarak kullanılabilir. Ancak bu iç yapıların bütün hacmi boyunca ve güvenilir bir anatomik yapıların tespit edilebilir bir numune için tespit olmayabilir. Bazı gruplar bir çift-bilge kayıt yaklaşım kullanılmış ve ardışık histoloji görüntüleri hatlarını ya da anatomik yapıları 16-18 kullanarak başka bir kayıt var. Referans resim kullanılmadan birbirine seri histoloji kısımlarının kayıt tarihi hata yaymak ve histoloji birimin gerçek şekli değişebilir. Pair-bilge kayıt yaklaşım görüntülerin yığını boyunca histoloji bölümlerinde ve iç yapılarının şekli tutarlılık dayanır; bu nedenle numunenin yoğun örnekleme gerektirenklinik örnekler için, örneğin belki her zaman mümkün değildir. Bu boru hattı biz histoloji hacim rekonstrüksiyonu 19 için referans görüntülerin bir dizi olarak blockface görüntüleri kullanın. Blockface görsel mikrotomu monte edildikten sonra parafin doku blokları alınır ve her bir bölüm kesilmeden önce. Böylece, bireysel seri bölümleri kesmek için hasar seri bölümleri 8,11,15 tesciline engel değildir. Biz, diğer gruplardan farklı bir şekilde blockface çekim. Optik blok yüz görüntüleri optik düzenli lens kullanırken genellikle oluşur varil ve perspektif bozulmalarını ortadan kaldırmak veya en aza indirmek için bir Telesentrik lens ile elde edilir. Bu normal bir lens kullanarak blockface görüntüleme gerçekleştirmek yayınlanan diğer yöntemlere göre önerilen yaklaşımın avantajlarından biridir. Görüntüler Tiss arasındaki kontrast, bloğun yüzeyi yansımasını kullanmak için hafif eğik bir açı ile alınırue ve parafin yüzeyi ve parafin yüzeyinin altında, derinlemesine doku gölge ortadan kaldırmak için. Bir fotoğrafik filtre, aynı zamanda blok yüzeyi ile kontrast 19 dengelemek için dokudan gelen ışığı polarize etmek için kullanılır. Döner mikrotom üzerindeki bloğun değiştirmesi için düzeltmek için, 2-3 delik blockface görüntülerde kolaylıkla saptanabilen bloğun köşelerinde, delinir. Bu deliklerin sentroidler blockface görüntüleri hizalamak için dönüm noktası tabanlı katı kaydı ile birlikte kullanılır.

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

Histoloji hacmi yeniden şekil 3B ve hücrelerden oluşur ölçekli yapılar düzeyinde bir organın hacim değişikliği üzerinde çalışmayı kolaylaştırmaktadır. Ayrıca, hacimsel tıbbi görüntüleme ve terapilerde yeni teknikleri ve algoritmalar araştırmak ve doğrulamak için kullanılabilir. Farklı organlarda 1,2,3 3D yüksek çözünürlüklü atlas oluşturma histoloji hacim rekonstrüksiyonu başka bir uygulamadır. Bu doku yapıları ve çeşitli hücresel özellikler arasındaki mekansal ilişkinin araştırmak için bir kaynak sağlar. Biz optik blockface görüntüleri kümesi kullanır histoloji hacim oluşturulması için bir görüntü kaydı yaklaşım sunuyoruz. Yeniden inşa histoloji cilt no bitkiler, post-processing kayıt hatası ile işlenmiş numune güvenilir bir şekli temsil eder. Iki fare meme bezlerinin Hematoksilen ve Eosin (H &amp; E) boyanan kesitler ed çıkarılan sınır noktalarını kullanarak karşılık gelen blockface görüntülere tescil edildihistoloji ve blockface görüntülerde numunenin Ges. Kayıt doğruluğu görsel olarak değerlendirilmiştir. Meme bezlerinin makro yapılar uyumu da görsel olarak yüksek çözünürlükte değerlendirildi. Bu çalışma, meme bezinin eksizyonu 3D histoloji hacim rekonstrüksiyonu kadar değişen, bu görüntü kayıt boru hattının farklı adımlar çizer. 2D histoloji görsel bölümlerin çiftleri arasındaki yapısal farkların ortaya koymakla birlikte, 3 boyutlu hacim histoloji şekli ve meme bezlerinin hacim farklılıkları görselleştirmek için olanağı sağlar.

Geleneksel mikroskopi teknikleri bir hatadır mikroskobik düzeyde bir organın anlayış bir defada bir alan-görünümü ile sınırlı olmasıdır. Hatta tüm slayt bölümlerini sağlamak &quot;total açıklama&quot; slaytlar, üç boyutlu bilgi sağlamak için başarısız. Bütün slayt, dinamik tarama teknolojileri, bütünüyle bir bölümünü görmek bizim yeteneği arttı, gelişimi ile ancak yapıları extrapolating 3D histoloji hacmi rekonstrüksiyon gerektirir. Daha Igfbp7 null...

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

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

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

3-Boyutlu Histoloji hacmi ve Fare meme bezlerinin Eğitim için onun Uygulama İmar

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

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

Research

JoVE Journal

Biology

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

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

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

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

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

Kertenkeleler Hareket Hız ve Yön Visual Hassasiyet Test

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

Biyoloji

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

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

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

Görev Yürüyüş Merdiven Basamak: Puanlama Sistemi ve Pratik Uygulama.

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

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

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

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

Zebra balığı Lens Nakli ve Göz Mutants Analizi Uygulaması

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

Mouse Mammary Epithelial Cells form Mammospheres During Lactogenic Differentiation

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

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

Fare Meme Epitel Hücreleri lactogenic Farklılaşma sırasında Mammospheres formu

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

Evaluation of Mammary Gland Development and Function in Mouse Models

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

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

Fare Modeller Meme Bezi Geliştirme ve Fonksiyon Değerlendirme

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

Intraductal Injection for Localized Drug Delivery to the Mouse Mammary Gland

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

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

Fare meme bezi Lokalize İlaç Teslim Intradüktal Enjeksiyon

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

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

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

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

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

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

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

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

İlköğretim Fare Osteoblastlar gelen osteositlere Kültürler Edinme Retinoik Asit Uygulama

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

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

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

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

Tümör sferoidlerin Yüksek throughput Görüntü Analizi: Otomatik ve Doğru küremsiler boyutunu ölçmek için kullanıcı-dostu Yazılım Uygulama

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

Measuring Pressure Volume Loops in the Mouse

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

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

Fare Basınç Hacim Döngüler Ölçme

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