AP Aiden, XR Bao, M. Brenner, D. Galas, W. Gosper, A. Jaffer, A. Melnikov, A. Miele, G. Giannoukos, C. Nusbaum, AJM Walhout; A. Kosmrlj tartışmalar ve kod için teşekkür ederim , L. Wood, K. Zeldovich tartışmaları, görselleştirme ile yardım ve L. Gaffney ve B. Wong. Fannie ve John Hertz Vakfı lisansüstü burs, Milli Savunma Bilimi ve Mühendisliği yüksek lisans burs, NSF lisansüstü burs, Ulusal Uzay Biyomedikal Araştırma Enstitüsü, hiçbir hibe tarafından desteklenir. Ulusal İnsan Genomu Araştırma Enstitüsü (NHGRI) (EL) T32 HG002295; i2b2 (Biyoloji ve Hastabaşı entegre Bilişim), Brigham ve Kadın Hastanesi (LAM) Biyomedikal Computing NIH destekli Merkezi; hibe yok. NHGRI HG003143 ve Keck Vakfı ayırt genç bilim adamı ödülü (JD). Ham ve eşlenmiş Hi-C dizisi veri GEO veritabanı (tevdi edilmiştir <a href="http://www.ncbi.nlm.nih.gov/geo/">www.ncbi.nlm.nih.gov/geo/</a> üyelik yok). GSE18199. Ek görselleştirme. <a href="http://hic.umassmed.edu">http://hic.umassmed.edu</a> .

<ol>
	<li>Lieberman-Aiden, E., Van Berkum, N. L., Williams, L., Imakaev, M., Ragoczy, T., Telling, A., Amit, I., Lajoie, B. R., Sabo, P. J., Dorschner, M. O., Sandstrom, R., Bernstein, B., Bender, M. A., Groudine, M., Gnirke, A., Stamatoyannopoulos, J., Mirny, L. A., Lander, E. S., Dekker, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+mapping+of+long-range+interactions+reveals+folding+principles+of+the+human+genome.">Comprehensive mapping of long-range interactions reveals folding principles of the human genome.</a> Science. 326, 289-293 (2009).</li><li>Kosak, S. T., Groudine, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Form+follows+function:+the+genomic+organization+of+cellular+differentiation.">Form follows function: the genomic organization of cellular differentiation.</a> Genes and Dev. 18, 1371-1384 (2004).</li><li>Misteli, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+the+sequence:+cellular+organization+of+genome+function.">Beyond the sequence: cellular organization of genome function.</a> Cell. 128, 787-800 (2007).</li><li>Dekker, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+Regulation+in+the+Third+Dimension.">Gene Regulation in the Third Dimension.</a> Science. 319, 1793-1794 (2008).</li><li>Cremer, T., Cremer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome+territories,+nuclear+architecture+and+gene+regulation+in+mammalian+cells.">Chromosome territories, nuclear architecture and gene regulation in mammalian cells.</a> Nat Rev Genet. 2, 292-301 (2001).</li><li>Sexton, T., Schober, H., Fraser, P., Gasser, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+regulation+through+nuclear+organization.">Gene regulation through nuclear organization.</a> Nat Struct and Mol Biol. 14, 1049-1055 (2007).</li><li>Dekker, J., Rippe, K., Dekker, M., Kleckner, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capturing+Chromosome+Conformation.">Capturing Chromosome Conformation.</a> Science. 295, 1306-1311 (2002).</li><li>Zhao, Z., Tavoosidana, G., Sj&#246;linder, M., G&#246;nd&#246;r, A., Mariano, P., Wang, S., Kanduri, C., Lezcano, M., Sandhu, K. S., Singh, U., Pant, V., Tiwari, V., Kurukuti, S., Ohlsson, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circular+chromosome+conformation+capture+(4C)+uncovers+extensive+networks+of+epigenetically+regulated+intra-+and+interchromosomal+interactions.">Circular chromosome conformation capture (4C) uncovers extensive networks of epigenetically regulated intra- and interchromosomal interactions.</a> Nat Genet. 38, 1341-1347 (2006).</li><li>Simonis, M., Klous, P., Splinter, E., Moshkin, Y., Willemsen, R., de Wit, E., van Steensel, B., de Laat, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+organization+of+active+and+inactive+chromatin+domains+uncovered+by+chromosome+conformation+capture-on-chip+(4C)+uncovers+extensive+networks+of+epigenetically+regulated+intra-+and+interchromosomal+interactions.">Nuclear organization of active and inactive chromatin domains uncovered by chromosome conformation capture-on-chip (4C) uncovers extensive networks of epigenetically regulated intra- and interchromosomal interactions.</a> Nat Genet. 38, 1348-1354 (2006).</li><li>Dostie, J., Richmond, T. A., Arnaout, R. A., Selzer, R. R., Lee, W. L., Honan, T. A., Rubio, E. D., Krumm, A., Lamb, J., Nusbaum, C., Green, R. D., Dekker, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome+Conformation+Capture+Carbon+Copy+(5C):+A+massively+parallel+solution+for+mapping+interactions+between+genomic+elements.">Chromosome Conformation Capture Carbon Copy (5C): A massively parallel solution for mapping interactions between genomic elements.</a> Genome Res. 16, 1299-1309 (2006).</li><li>Miele, A., Dekker, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+Cis-+and+Trans+Chromatin+Interaction+Networks+Using+Chromosome+Conformation+Capture+(3C).">Mapping Cis- and Trans Chromatin Interaction Networks Using Chromosome Conformation Capture (3C).</a> Methods Mol Biol. 464, 105-121 (2009).</li><li>Maccallum, I., Przybylski, D., Gnerre, S., Burton, J., Shlyakhter, I., Gnirke, A., Malek, J., McKernan, K., Ranade, S., Shea, T. 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Hi-C yöntemi geçici patent (no. 61/100, 151), inceleme altında.

Biz kromatin etkileşimleri, tarafsız bir genom şekilde haritalama, genomun 3 boyutlu mimari okuyan bir yöntem mevcut. Önceki çalışma dışında bu teknoloji ayarlar en kritik deneysel adım künt uç ligasyonu önce çapraz parçalarının biter kısıtlama biotinlenmiş nükleotidlerin eklenmesi. Bu adımı başarıyla derin tüm ligasyonu kavşak sıralama sağlar ve Hi-C, kapsamı ve güç verir. Okunma sayısı sonuçta etkileşim haritalar kararı belirleyecektir. Burada, insan genomu için 1 Mb etkileşim harita ~ 30 milyon alignable okuma kullanarak sunulmaktadır. N bir faktör &#39;tüm amacı&#39; çözümü artırmak amacıyla, okur sayısı n 2 bir faktör artış olmalıdır. Hi-C tekniği kolayca kütüphane nesil sonra hibrid yakalama (genomun belirli bölümlerine hedef) ve bağlanmasından sonra kromatin immunoprecipitation (spesifik protein ile ilişkili bölgelerde kromatin çevreyi araştırmak) gibi diğer teknikleri ile kombine edilebilir.

Erratum:

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<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>Protease inhibitors</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P8340-5ml</td>
<td>Step 1.2</td>
</tr>
<tr>
<td>biotin-14-dCTP</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>19518-018</td>
<td>Step 1.6</td>
</tr>
<tr>
<td>Klenow</td>
<td>&nbsp;</td>
<td>NEB</td>
<td>M0210</td>
<td>Steps 1.6 and 2.2</td>
</tr>
<tr>
<td>T4 DNA ligase</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15224</td>
<td>Step 1.9</td>
</tr>
<tr>
<td>T4 DNA polymerase</td>
<td>&nbsp;</td>
<td>NEB</td>
<td>M0203</td>
<td>Steps 1.17 and 2.2</td>
</tr>
<tr>
<td>10x ligation buffer</td>
<td>&nbsp;</td>
<td>NEB</td>
<td>B0202</td>
<td>Steps 2.2 and 3.4</td>
</tr>
<tr>
<td>T4 PNK</td>
<td>&nbsp;</td>
<td>NEB</td>
<td>M0201</td>
<td>Step 2.2</td>
</tr>
<tr>
<td>Klenow (exo-)</td>
<td>&nbsp;</td>
<td>NEB</td>
<td>M0212</td>
<td>Step 2.3</td>
</tr>
<tr>
<td>Dynabeads MyOne Streptavin C1 Beads</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>650.01</td>
<td>Step 3.2</td>
</tr>
<tr>
<td>T4 DNA ligase HC</td>
<td>&nbsp;</td>
<td>Enzymatics</td>
<td>L603-HC-L</td>
<td>Step 3.5</td>
</tr>
<tr>
<td>Phusion HF mastermix</td>
<td>&nbsp;</td>
<td>NEB</td>
<td>F531</td>
<td>Step 3.8</td>
</tr>
<tr>
<td>Ampure beads</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>A2915</td>
<td>Step 3.9</td>
</tr>		</tbody>
		</table>
		</div>

hi-c: a method to study the three-dimensional architecture of genomes.

Kromozom üç boyutlu katlama genom compartmentalizes ve yakın mekansal yakınlığı 2-6 içine rehberleri ve arttırıcılar gibi uzak fonksiyonel elemanları, getirebilir. Kromozom organizasyon ve genom aktivitesi arasında ilişki Deşifre transkripsiyon ve replikasyon gibi, genomik süreçleri anlamada yardımcı olacaktır. Ancak, kromozom kat hakkında çok az bilinir. Mikroskopi lokusların aynı anda yüksek çözünürlükte çok sayıda ayırt etmek mümkün. Bugüne kadar, kromozom konformasyon yakalama (3C) ve sonraki uyarlamalar kullanarak kromozom etkileşimleri tespit genom çalışmaları imkansız 7-10 yapma, hedef lokusların bir dizi seçim gereklidir. Biz Hi-C, 3C tarafsız, genom moda uzun menzilli etkileşimleri belirleme yeteneğine sahip bir uzantısı. Hi-C, hücreleri etkileşim lokusların kovalent DNA-protein çapraz bağlantılar yoluyla birbirine bağlı olmayı neden, formaldehit ile sabittir. DNA daha sonra bir restriksiyon enzimi ile parçalanmış, bu lokuslar bağlı kalır. 5 &#39;çıkıntılar yanında, künt uç ligasyonu, çapraz bağlı DNA parçaları arasında ligasyonu olayları lehine seyreltik koşullar altında yapılır doldurulur biotinlenmiş kalıntı kurulmuştur. Ligasyonu ürünlerin genom kütüphane Bu sonuç, çekirdeğinde birbirine yakın başlangıçta parçaları çiftleri karşılık. Her ligasyonu ürün kavşak yerinde biotin ile işaretlenmiş. Kütüphane makaslanmış ve kavşak streptavidin boncuk aşağı çekti. Saflaştırılmış kavşak sonradan etkileşim parçalarının bir katalog, yüksek verimli bir sequencer kullanarak analiz edilebilir. Doğrudan temas matris analizi, kromozom topraklarının varlığı ve küçük zengin gen-kromozom tercihli dernek olarak, genomik organizasyonu sayısız özellikleri ortaya koymaktadır. Korelasyon analizinde, insan genomunun iki bölmeye ayrılmış olduğunu gösteren iletişim matris uygulanabilir: kapalı, ulaşılmaz ve etkin kromatin bölgeleri içeren, açık, ulaşılabilir ve aktif kromatin ve daha yoğun bir bölmesi içeren daha az yoğun paketlenmiş bölmesi. Son olarak, teorik türevleri ve hesaplama simülasyonları ile birleştiğinde topluluğu iletişim matris analizi, Hi-C megabase ölçekte bir fraktal küreciğin konformasyon ile tutarlı özellikler gösterdiği saptandı.

Watch this Scientific Journal Video about Hi-C: A Method to Study the Three-dimensional Architecture of Genomes. at JoVE.com

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

Hi-C yöntemi kromatin etkileşimler (1) tarafsız, genom kimlik sağlar. Hi-C çiftler yakınlık ligasyonu ve güçlü paralel sıralama. Ortaya çıkan veriler farklı ölçeklerde genomik mimarlık eğitimi için kullanılabilir: ilk sonuçlar, kromozom toprakları, segregasyon, açık ve kapalı kromatin ve megabase ölçekte kromatin yapısı gibi özellikleri tespit.

Hi-C: genomların Üç boyutlu Mimari Eğitim Bir Yöntem.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

hi-c-a-method-to-study-the-three-dimensional-architecture-of-genomes

Research

JoVE Journal

Biology

408.3K Görüntüleme.  University of Massachusetts Medical School. Hi-C yöntemi kromatin etkileşimler (1) tarafsız, genom kimlik sağlar. Hi-C çiftler yakınlık ligasyonu ve güçlü paralel sıralama. Ortaya çıkan veriler farklı ölçeklerde genomik mimarlık eğitimi için kullanılabilir: ilk sonuçlar, kromozom toprakları, segregasyon, açık ve kapalı kromatin ve megabase ölçekte kromatin yapısı gibi özellikleri tespit.

Video: Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

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

A high-throughput method to globally study the organelle morphology in S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help achieve an comprehensive understanding of molecular pathways. In Saccharomyces cerevisiae, with the development of the non-essential gene deletion array, we can globally study the morphology of different organelles like the endoplasmic reticulum (ER) and the mitochondria using GFP (or variant)-markers in different gene backgrounds. However, incorporating GFP markers in each single mutant individually is a labor-intensive process. Here, we describe a procedure that is routinely used in our laboratory. By using a robotic system to handle high-density yeast arrays and drug selection techniques, we can significantly shorten the time required and improve reproducibility. In brief, we cross a GFP-tagged mitochondrial marker (Apc1-GFP) to a high-density array of 4,672 nonessential gene deletion mutants by robotic replica pinning.  Through diploid selection, sporulation, germination and dual marker selection, we recover both alleles. As a result, each haploid single mutant contains Apc1-GFP incorporated at its genomic locus. Now, we can study the morphology of mitochondria in all non-essential mutant background. Using this high-throughput approach, we can conveniently study and delineate the pathways and genes involved in the inheritance and the formation of organelles in a genome-wide setting.

GFP-fusion proteins are widely used to visualize organelles by confocal microscopy. However, screening for mutations that affect the morphology of organelles generally requires individual mutagenesis and is time consuming. Here, we demonstrate a method to simultaneously incorporate organelle-GFP markers in almost 5,000 non-essential genes in yeast.

S. cerevisiae &quot;adlı organel morfolojisi küresel çalışma yüksek verimli bir yöntem

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help ...

Using High Resolution Computed Tomography to Visualize the Three Dimensional Structure and Function of Plant Vasculature

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being used to evaluate the structure and function of plant xylem network in three dimensions (3D) (e.g. Brodersen et al. 2010; 2011; 2012a,b). HRCT imaging is based on the same principles as medical CT systems, but a high intensity synchrotron x-ray source results in higher spatial resolution and decreased image acquisition time. Here, we demonstrate in detail how synchrotron-based HRCT (performed at the Advanced Light Source-LBNL Berkeley, CA, USA) in combination with Avizo software (VSG Inc., Burlington, MA, USA) is being used to explore plant xylem in excised tissue and living plants. This new imaging tool allows users to move beyond traditional static, 2D light or electron micrographs and study samples using virtual serial sections in any plane. An infinite number of slices in any orientation can be made on the same sample, a feature that is physically impossible using traditional microscopy methods.
Results demonstrate that HRCT can be applied to both herbaceous and woody plant species, and a range of plant organs (i.e. leaves, petioles, stems, trunks, roots). Figures presented here help demonstrate both a range of representative plant vascular anatomy and the type of detail extracted from HRCT datasets, including scans for coast redwood (Sequoia sempervirens), walnut (Juglans spp.), oak (Quercus spp.), and maple (Acer spp.) tree saplings to sunflowers (Helianthus annuus), grapevines (Vitis spp.), and ferns (Pteridium aquilinum and Woodwardia fimbriata). Excised and dried samples from woody species are easiest to scan and typically yield the best images. However, recent improvements (i.e. more rapid scans and sample stabilization) have made it possible to use this visualization technique on green tissues (e.g. petioles) and in living plants. On occasion some shrinkage of hydrated green plant tissues will cause images to blur and methods to avoid these issues are described. These recent advances with HRCT provide promising new insights into plant vascular function.

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique that can be used to study the structure and function of plant vasculature in 3D. We demonstrate how HRCT facilitates exploration of xylem networks across a wide range of plant tissues and species.

Bitki damarları ve Üç Boyutlu Yapı ve Fonksiyon Visualize&#39;a Yüksek Çözünürlüklü Bilgisayarlı Tomografi Kullanımı

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being ...

Slide Preparation Method to Preserve Three-dimensional Chromatin Architecture of Testicular Germ Cells

During testicular germ cell differentiation, the structure of nuclear chromatin dynamically changes. The following describes a method designed to preserve the three-dimensional chromatin arrangement of testicular germ cells found in mice; this method has been termed as the three-dimensional (3D) slide method. In this method, testicular tubules are directly treated with a permeabilization step that removes cytoplasmic material, followed by a fixation step that fixes nuclear materials. Tubules are then dissociated, the cell suspension is cytospun, and cells adhere to slides. This method improves sensitivity towards detection of subnuclear structures and is applicable for immunofluorescence, DNA, and RNA fluorescence in situ hybridization (FISH) and the combination of these detection methods. As an example of a possible application of the 3D slide method, a Cot-1 RNA FISH is shown to detect nascent RNAs. The 3D slide method will facilitate the detailed examination of spatial relationships between chromatin structure, DNA, and RNA during testicular germ cell differentiation.

The material here describes a method developed to preserve the three-dimensional chromatin structure of testicular germ cells. This has been termed the three-dimensional (3D) slide method. This method improves sensitivity for detection of subnuclear structures and is applicable for immunofluorescence, DNA, and RNA fluorescence in situ hybridization (FISH).

Testis Germ Hücrelerinin Üç Boyutlu Kromatin Mimarisini Korumak için Slayt Hazırlama Yöntemi

During testicular germ cell differentiation, the structure of nuclear chromatin dynamically changes. The following describes a method designed to preserve ...

FRET Imaging in Three-dimensional Hydrogels

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

F&#246;rster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.

Üç boyutlu Hidrojeller içinde Görüntüleme FRET

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...

Using a Whole-mount Immunohistochemical Method to Study the Innervation of the Biliary Tract in Suncus murinus

This work describes the whole-mount immunohistochemistry staining method in detail, using neurofilament protein antibody to label the innervation of the biliary tract in Suncus murinus (S. murinus&#160;). First, the specimen was dissected from S. murinus and fixed in 4% paraformaldehyde (PFA). Second, an enzymatic treatment and potential endogenous peroxidase inactivation were performed. The specimen was then exposed to the primary antibody, anti-neurofilament protein antibody, for 3-6 days. It was then incubated with the secondary antibody conjugated with horseradish peroxidase. The color reaction was revealed by reacting the specimen with a 3,3&#39;-diaminobenzidine (DAB) substrate. This method can be applied to analyze the innervation of all visceral organs. Furthermore, this protocol can also be adapted to test other neuronal antibodies, but optimization of the antibodies should be done first. This method was originally introduced by Kuratani and Tanaka1,2,3.

Using a Whole-mount Immunohistochemical Method to Study the Innervation of the Biliary Tract in Suncus murinus

A whole-mount immunohistochemical approach, to visualize neurofilament protein expression in the extrahepatic biliary tract in Suncus murinus. is presented here. This protocol can be used to analyze the innervation of all visceral organs in S. murinus or other species.

Safra Yolunun İnternalasyonunu İncelemek İçin Bir Tam Montajlı İmmünohistokimyasal Yöntem Kullanmak<em&gt; Suncus murinus</em

This work describes the whole-mount immunohistochemistry staining method in detail, using neurofilament protein antibody to label the innervation of the ...

Three-dimensional Reconstruction of the Vascular Architecture of the Passive CLARITY-cleared Mouse Ovary

The ovary is the main organ of the female reproductive system and is essential for the production of female gametes and for controlling the endocrine system, but the complex structural relationships and three-dimensional (3D) vasculature architectures of the ovary are not well described. In order to visualize the 3D connections and architecture of blood vessels in the intact ovary, the first important step is to make the ovary optically clear. In order to avoid tissue shrinkage, we used the hydrogel fixation-based passive CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/ Immunostaining/In situ-hybridization-compatible Tissue Hydrogel) protocol method to clear an intact ovary. Immunostaining, advanced multiphoton confocal microscopy, and 3D image-reconstructions were then used for the visualization of ovarian vessels and follicular capillaries. Using this approach, we showed a significant positive correlation (P &#60;0.01) between the length of the follicular capillaries and volume of the follicular wall.

Here we present an adaptation of the passive CLARITY and 3D reconstruction method for visualization of the ovarian vasculature and follicular capillaries in intact mouse ovaries.

Pasif AÇIKLIĞI temizlenmiş Mouse ovarium vasküler mimarisinin üç boyutlu yeniden yapılandırma

The ovary is the main organ of the female reproductive system and is essential for the production of female gametes and for controlling the endocrine ...

Combining Chemical Cross-linking and Mass Spectrometry of Intact Protein Complexes to Study the Architecture of Multi-subunit Protein Assemblies

Proteins interact with their ligands to form active and dynamic assemblies which carry out various cellular functions. Elucidating these interactions is therefore fundamental for the understanding of cellular processes. However, many protein complexes are dynamic assemblies and are not accessible by conventional structural techniques. Mass spectrometry contributes to the structural investigation of these assemblies, and particularly the combination of various mass spectrometric techniques delivers valuable insights into their structural arrangement.
In this article, we describe the application and combination of two complementary mass spectrometric techniques, namely chemical cross-linking coupled with mass spectrometry and native mass spectrometry. Chemical cross-linking involves the covalent linkage of amino acids in close proximity by using chemical reagents. After digestion with proteases, cross-linked di-peptides are identified by mass spectrometry and protein interactions sites are uncovered. Native mass spectrometry on the other hand is the analysis of intact protein assemblies in the gas phase of a mass spectrometer. It reveals protein stoichiometries as well as protein and ligand interactions. Both techniques therefore deliver complementary information on the structure of protein-ligand assemblies and their combination proved powerful in previous studies.

The architecture of protein complexes is essential for their function. Combining various mass spectrometric techniques proved powerful to study their assembly. We provide protocols for chemical cross-linking and native mass spectrometry and show how these complementary techniques help to elucidate the architecture of multi-subunit protein assemblies.

Kimyasal Cross-linking ve çoklu alt birim Protein derlemeler mimarlık okumak için olduğu gibi Protein kompleksleri kütle spektrometresi birleştirme

Proteins interact with their ligands to form active and dynamic assemblies which carry out various cellular functions. Elucidating these interactions is ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipo-Clear: Bir doku Yöntem yağ dokusu üç boyutlu görüntüleme için temizlenmesi

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...

Quadruple-Checkerboard: A Modification of the Three-Dimensional Checkerboard for Studying Drug Combinations

The concept of drug-combination therapy is becoming very important mainly with the drastic increase in resistance to drugs. The Quadruple&#160;checkerboard, also called the Q-checkerboard, aims at maximizing the number of possible combinations that can be obtained between four drugs in one experiment to minimize the time and work needed to accomplish the same results with other protocols. This protocol is based on the simple micro dilution technique where the drugs are diluted and combined together in several 96-well plates.
In the first set of 96-well plates, Muller-Hinton broth is added followed by the first required drug (e.g., Cefotaxime here) to serially dilute it. After the first step is done, another set of 96-well plates is used to dilute the second drug (e.g., Amikaci), which will be transferred by removing a specific volume of drug 2 and put in the corresponding wells in the first set of 96-well plates that contains drug one. The third step is done by adding the required concentrations of the third drug (e.g., Levofloxacin), to the appropriate plates in the initial set containing combination of drug 1 and 2. The fourth step is done by adding the required concentrations of the fourth drug (e.g., Trimethoprim-sulfamethoxazol) into the appropriate plates in the first set. Then, E. coli ESBL bacterial inoculum will be prepared and added.
This method is important to evaluate all the possible combinations and has a wider range of possibilities to be tested furthermore for in vivo testing. Despite being a tiring technique requiring a lot of focus, the results are remarkable and time saving where a lot of combinations can be tested in a single experiment.

This protocol describes how to study all possible combinations that can be obtained between four drugs in one single experiment. This method is based on the standard 96-well plate micro dilution assay and the calculation of fractional inhibitory concentrations (FICs) to evaluate the results.