למחברים אין תודות.

כל הניסויים הניסויים אושרו על ידי אוניברסיטת טוקיו מטרופולין מוסדיים טיפול בבעלי חיים ועדת שימוש (IACUC). החיות שוכנו ומטופלות בהתאם למדריך לטיפול ולשימוש בחיות מעבדה ובמדריך לטיפול ולשימוש בבעלי חיים ניסיוניים של המועצה הקנדית לטיפול בבעלי חיים. השתמשנו בבעלי חיים וגידלנו במעבדה למורפולוגיה תפקודית, המחלקה למדעי הבריאות של הגבול, אוניברסיטת מטרופולין של טוקיו, יפן. 1. טיפול בבעלי חיים ודיור <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> השג 7 ס מורינוס (3 נקבות ו 4 זכרים במשקל 70-90 גרם) ממושבה ריבוי סגורה. </li><li style=";text-align:right;direction:rtl"> לכלוב את ס מורינוס בנפרד לאחר הגמילה (20 ד לאחר הלידה) בכלובי פלסטיק מצוידים תיבת קינון עץ המכילים רצועות נייר. שמור אותם בחדר חיות מותנה באופן קונבנציונלי: 23-27 ° C, ללא בקרת לחות, ו 12:12 שעה אור: מחזור כהה. ספק אספקהIal pellets ו מים המודעה libitum. </li></ol> 2. הכנת רקמות <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> כדי להכין 4% (w / v) paraformaldehyde (PFA), להמיס 40 גרם של PFA ב 1000 מ&quot;ל של 0.01 M זרחן שנאגרו מלוחים (PBS, pH 7.4). בתוך ארון אדים, מערבבים באמצעות מערבולת עד הפתרון ברור. חנות 4% PFA O / N ב 4 ° C. זהירות: ללבוש המתאים מגן אישי ציוד (PPE) בעת הטיפול PFA. </li><li style=";text-align:right;direction:rtl"> כדי perfuse ולתקן את החיה, להרדים אותו עם אתר ולאחר מכן לתת לו זריקה intraperitoneal של somnopentyl (pentobarbital נתרן, 0.6 מ&quot;ל / ק&quot;ג bodyweight). </li><li style=";text-align:right;direction:rtl"> לאחר החיה מסומנת לחלוטין, לפתוח את חלל הבטן עם אזמל, יצירת חתך הבטן קו האמצע 3 ס&quot;מ. בעוד incising נחות הווריד הנבוב עבור exsanguinating, להכניס קטטר retrogradely לתוך אבי העורקים הבטן ברמה מיד מעל ההתפצלות של עורק זה לתוך העורקים השכיח המשותף. </li><li style=";text-align:right;direction:rtl"> להזריק somnopentyl (pentobarbital כךDium, 1.0 מ&quot;ל / ק&quot;ג bodyweight). לאחר המתת חסד מלאה, perfuse עם 0.01 M PBS (pH 7.4) ולאחר מכן עם PFA 4% בארון קטר. </li><li style=";text-align:right;direction:rtl"> לאחר זלוף, להזריק 2-3 מ&quot;ל של לטקס נייטרן לבן (לדלל לטקס נייטרן לבן 3: 2 עם מים מזוקקים (DW)) לתייג את כלי הדם. </li><li style=";text-align:right;direction:rtl"> לחלץ את איברי הבטן, כולל הכבד, כיס המרה, צינור המרה משותף, תריסריון, לבלב וגוש, עם עצבים הקשורים וכלי. </li><li style=";text-align:right;direction:rtl"> להזריק כ 0.1 מ&quot;ל של לטקס נייטרן כחול (להוסיף טיפה של דיו כחול ל 10 מ&quot;ל של דיאטן לטקס נייטרן לבן מדולל) כדי כיס המרה כדי התווית מערכת המרינה. </li><li style=";text-align:right;direction:rtl"> לאחר תיקון לילה עם PFA 4% ב 4 ° C עבור immunostaining הר כולו. זהירות: PFA הוא רעיל. הימנע טיפול PFA ישירות. PFA יש להשתמש בארון קטר. </li></ol> 3. שלמה הר אימונוהיסטוכימיה הערה: לאורך הפרוטוקול, כולל במהלך כביסה, הדגירה נוגדנים, צבע, thE specimen חייב להישאר על nutator, בעדינות נדנדה ב RT או 4 ° C. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> יום 1: טיפול אנזימטי ו inactivation פוטנציאל peroxidase אנדוגני <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> שטפו את הדגימה מוכנה עם DW 4x עבור 1 שעות כל ב RT ב בקבוקון בגודל מתאים. רוק את הדגימה בעדינות על nutator כדי להסיר את PFA. הימנע נזק הדגימה בעת החלפת פתרונות. </li><li style=";text-align:right;direction:rtl"> כדי להכין 1% (w / v) חומצה אורתופריאודית, להמיס 1 גרם של חומצה אורתופריאודלית ב 100 מ&quot;ל של DW. </li><li style=";text-align:right;direction:rtl"> דגירה הדגימה של 1% אורטופרידיום חומצה במשך 20 דקות ב RT כדי למנוע כל תגובה peroxidase מהותי. </li><li style=";text-align:right;direction:rtl"> הכן 0.004% (w / v) papain על ידי המסת 0.004 גרם של papain ב 100 מ&quot;ל של 0.025 mol / L Tris-HCl חיץ (pH 7.6). </li><li style=";text-align:right;direction:rtl"> שטפו את הדגימה עם DW במשך 10 דקות ב RT. </li><li style=";text-align:right;direction:rtl"> דגירה הדגימה של פאפאין 0.004% מוכן טרי עבור 2 שעות ב 37 מעלות צלזיוס באמבט טמפרטורה קבוע עם נדנדה עדינה. </li><li style=";text-align:right;direction:rtl"> לשטוף את הדגימה עם DW עבור 50-60 דקות ב RT. </li><li style=";text-align:right;direction:rtl"> חנות הדגימה 4% PFA ב 4 ° CO / N. </li></ol></li><li style=";text-align:right;direction:rtl"> יום 2: טיפול אנזימטי <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> שטפו את הדגימה המאוחסנת עם DW 4 פעמים במשך שעה 1 ב RT. </li><li style=";text-align:right;direction:rtl"> דגירה הדגימה של פאפאין 0.004% מוכן טרי עבור 2 שעות ב 37 מעלות צלזיוס באמבט טמפרטורה קבוע עם נדנדה עדינה. </li><li style=";text-align:right;direction:rtl"> שטפו את הדגימה עם DW עבור 50-60 דקות ב RT. חנות הדגימה 4% PFA ב 4 ° CO / N. </li></ol></li><li style=";text-align:right;direction:rtl"> יום 3: הקפאת הטיפול <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> שטפו את הדגימה המאוחסנת עם DW 4x עבור 1 שעות כל ב RT, כאמור. </li><li style=";text-align:right;direction:rtl"> הכינו 2.5% (w / v), 5% (w / v) ו -10% (w / v) סוכרוז על ידי המסת 2.5 גרם, 5 גרם, 10 גרם סוכרוז, בהתאמה, ב 100 מ&quot;ל של 0.01 M PBS ( PH 7.4). </li><li style=";text-align:right;direction:rtl"> לטבול את הדגימה ב 2.5% (w / v), 5% (w / v), ו 10% (w / v) סוכרוז במשך 30 דקות כל אחד. </li><li style=";text-align:right;direction:rtl"> להקפיא את הדגימה ב -20 מעלות צלזיוס למשך 30 - 60 דקות ולאחר מכן להפשיר לחלוטין אותו RT; לחזור על מחזור 3x. </li><li style=";text-align:right;direction:rtl"> חלק עליוןReare 2% Triton X-100 (v / v), להוסיף 2 מ&quot;ל של טריטון X-100 ל 100 מ&quot;ל של 0.01 M PBS (pH 7.4). </li><li style=";text-align:right;direction:rtl"> חנות הדגימה 2% Triton X-100 ב 4 ° CO / N. </li></ol></li><li style=";text-align:right;direction:rtl"> יום 4: הדגירה נוגדן ראשוני <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכן את הפתרון דילול של הנוגדן העיקרי על ידי המסת 0.2 גרם של אלבומין בסרום שור (BSA), 0.3 מ&quot;ל של טריטון X-100, ו 0.1 גרם של אזיד נתרן ב 100 מ&quot;ל של 0.01 M PBS (pH 7.4). זהירות: אזיד הנתרן הוא רעיל מאוד. שאיפה ונוגע יש להימנע. ללבוש PPE המתאים. </li><li style=";text-align:right;direction:rtl"> לדלל את הנוגדן העיקרי (NFP) 1: 600 במאגר דילול לעיל (שלב 3.4.1). דגירה הדגימה עם נוגדן NFP ב 4 מעלות צלזיוס במשך 3-6 ימים, שמירה על הדגימה בעדינות נדנדה על nutator. דגימות גדולות יותר ידרשו פעמים הדגירה גדל. </li></ol></li><li style=";text-align:right;direction:rtl"> הדגירה נוגדן משני <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לשטוף את הדגימה PBS 4x עבור 1 שעות כל ב RT כדי להסיר נוגדן העיקרי מאוגד. </lI <li style=";text-align:right;direction:rtl"> הכן את פתרון דילול הנוגדנים משני על ידי המסת 0.2 גרם של BSA ו 0.3 מ&quot;ל של טריטון X-100 ב 100 מ&quot;ל של 0.01 M PBS (pH 7.4). </li><li style=";text-align:right;direction:rtl"> לדלל את הנוגדנים המשניים (peroxidase- מצומדות, זיקה מטוהרים כבשים אנטי עכבר IgG-HRP) 1: 600 במאגר דילול (שלב 3.5.2). דגירה הדגימה עם נוגדנים משני ב 4 מעלות צלזיוס במשך 3 ד, ולשמור על הדגימה נדנדה בעדינות על nutator. </li></ol></li><li style=";text-align:right;direction:rtl"> צבע <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכן DAB פתרון צבע על ידי הוספת 0.002 גרם של 3,3&#39;-diaminobenzidine tetrahydrochloride (DAB) ל 100 מ&quot;ל של 0.05 מ&quot;ל / L Tris-HCl חיץ (pH 7.6) תחת ארון אדים, שמירה על הפתרון בחושך. </li><li style=";text-align:right;direction:rtl"> שטפו את הדגימות ב RT ב PBS 4x עבור 1 שעות כל אחד. </li><li style=";text-align:right;direction:rtl"> הוסף 10 μL של 30% H 2 O 2 ב טריים מוכן DAB פתרון צבע ו דגירה עם הפתרון ב 4 ° CO / N או במשך 3 ד בעוד נדנדה בעדינות על הנוטאטור. </li><li style=";text-align:right;direction:rtl"> עצור את התגובה על ידי פלהCing הדגימה PBS עם 0.04% Azide נתרן כאשר עוצמת מכתים אופטימלי הוא הגיע. הערה: הימנע משימוש של אזיד הנתרן עד שלב זה, כפי שהוא מעכב peroxidase חזרת. </li></ol></li><li style=";text-align:right;direction:rtl"> הדמיה של הרקמה המוכתמת <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> בזהירות להעביר את הדגימה לצלחת זכוכית פטרי (5 ס&quot;מ גבוה, 10 ס&quot;מ קוטר) המכיל PBS. לכידת שלם הר תמונות באמצעות מצלמה רכוב על stereomicroscope. הערה: כל הר רקמת מוכתם צריך להיות צילמו בעוד שקוע לחלוטין PBS על צלחת זכוכית שקופה. </li></ol></li></ol>

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המחברים אינם מכריזים על ניגודי אינטרסים.

עבודה זו מתארת ​​את הליך הניסוי להדמיה של העצבון של דרכי המרה המרגיעה באמצעות נוגדנים נגד חלבון neurofilament. פרוטוקול זה הותאם מן הפרוטוקול המתואר על ידי Kuratani ו Tanaka 1 , 2 , 3 . עבור ניסו...

הבית מושק זועק , סונקוס murinus , שייך סדר Insectivora ואת Soricidae המשפחה. יונק זעיר זה מופץ ברחבי דרום מזרח אסיה ומתגורר בתים ואזורי עשב הממוקמים ליד היישוב האנושי או עטים בקר 4 . מין זה מציג מאפיינים מורפולוגיים כלליים דומים יותר לבני אדם מאשר לבעלי מעבדה אחרים, כגון עכבר, חולדה ושפן. בעבר, כל ההר immunostaining עם סמן נוירון היקפי עבור חלבון ne Murphus neurofilament (NFP) שימש התווית העצבים ההיקפיים בלבלב 6 , הפודילה המרכזית של התריסריון 7 , pylorus 8 , gallbladder 5 , ו exthepatic דרכי השתן 9 . NFP הוא קומפלקס macromolecular כי הוא חלק cytoskeleton העצבית בוגרת. נוירופילמנט הקשורות חלבוןאינטראקציה בין NFP לבין הזיגוזום. הן פונקציה האנזים ואת המבנה של חלבונים מקשר מוסדרים על ידי NFP 10 . מתחם NFP מורכב משלושה פוליפפטידים: NF-L (70 kDa), NF-M (150-160 kDa) ו- NF-H (200 kDa). NFP ניתן למצוא אקסונים נוירונים הן במערכת העצבים המרכזית והפריפריה. נוגדן NFP אנטי אנושי הוכח לתווית אקסונים של מערכת העצבים המרכזית והפריפלית. מחקרים אנטומיים ואלקטרופיזיולוגיים הוכיחו כי הסוגר, כיס המרה, ודרכי העיכול הפרוקסימלי מחוברים 11 , 12 , 13 , 14 . עם זאת, התכונות המורפולוגיות של חיבורים העצבית שלהם עדיין לא הוגדרו. עבודה זו מדגימה את השימוש של שיטת הר שלמה immunohistochemical מכתים את התווית של העצבנות של ס מורינוס bilIary עם נוגדנים NFP.

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			<td height="42" style="height:42px;width:216px;">3,3&#39;-diaminobenzidine tetrahydrochloride (DAB)</td>
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			<td height="21" style="height:21px;width:216px;">imidazole</td>
			<td style="width:71px;">Sigma</td>
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using a whole-mount immunohistochemical method to study the innervation of the biliary tract in suncus murinus

עבודה זו מתארת ​​את שיטת הרמוניה של המערכת החיסונית בכללותה, תוך שימוש בנוגדן נוירופילמנט לחלבון כדי לתייג את העצבון של דרכי המרה במעי הגס של סונקוס ( S. Murinus &#160; ). ראשית, הדגימה גזור מ מ &#39;מנוס ו קבוע ב paraformaldehyde 4% (PFA). שנית, טיפול אנזימטי ואקטיביזם peroxidase אנדוגני פוטנציאלי בוצעו. הדגימה נחשפה אז נוגדן ראשוני, נוגדן נוגדנים נוירופילמנט, במשך 3-6 ימים. זה היה מודגרות אז עם נוגדנים משני מצומדות עם חזרת peroxidase. התגובה הצבעונית התגלה על ידי תגובה הדגימה עם המצע של 3,3 &#39;-diaminobenzidine (DAB). שיטה זו יכולה להיות מיושמת כדי לנתח את העצבון של כל האיברים הקרביים. יתר על כן, פרוטוקול זה יכול גם להיות מותאם לבדוק נוגדנים נוירונים אחרים, אבל אופטימיזציה של antibodiEs צריך להיעשות הראשון. שיטה זו הוצגה במקור על ידי Kuratani ו Tanaka 1 , 2 , 3 .

<html> איורים 1 - 4 מראים תוצאות טיפוסיות עבור סיבי עצב NFP חיובי במערכת המעי הרגיז ב S. Murinus . נוגדן נגד NFP שכותרתו את העצבון בתמונה כולה של דרכי מרה מרהיב ( איור 1 ), כיס המרה ( איור 2 ), צינור המרה העליונה ( ...

Watch this Scientific Journal Video about Using a Whole-mount Immunohistochemical Method to Study the Innervation of the Biliary Tract in Suncus murinus at JoVE.com

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

גישה הר שלמה אימונוהיסטוכימי, כדי לחזות ביטוי חלבון neurofilament במערכת מרה מרהיב סונקוס murinus. מוצג כאן. פרוטוקול זה ניתן להשתמש כדי לנתח את העצבון של כל האיברים הקרביים בס מורינוס או מינים אחרים.

באמצעות שיטת הר שלמה Immunohistochemical ללמוד את שימור של דרכי המרה<em&gt; סונקוס מורן</em

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

using-whole-mount-immunohistochemical-method-to-study-innervation

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

6.7K Views.  Tokyo Metropolitan University.  גישה הר שלמה אימונוהיסטוכימי, כדי לחזות ביטוי חלבון neurofilament במערכת מרה מרהיב סונקוס murinus. מוצג כאן. פרוטוקול זה ניתן להשתמש כדי לנתח את העצבון של כל האיברים הקרביים בס מורינוס או מינים אחרים. 

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

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.

בדיקת רגישות חזותית על מהירות ועל כיוון התנועה של לטאות

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

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

הר שלם כפול באתרו הכלאה של עוברים צ&#39;יק מוקדם

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Method for Whole Mount Antibody Staining in Chick

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

שיטת מכתים נוגדן שלם הר ב צ&#39;יק

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red 1,2. This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) 3. The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy 3,4. One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein 5. When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. We include a propidium iodide nuclear counter-stain to highlight tissue organization.

דג הזברה שלמים הר ברזולוציה גבוהה פעמיים פלורסנט באתרו הכלאה

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

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

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

Hi-C: שיטה לחקר ארכיטקטורה תלת ממדי של הגנום.

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

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Whole mount in situ hybridization (WISH) is a common technique in molecular biology laboratories used to study gene expression through the localization of specific mRNA transcripts within whole mount specimen. This technique (adapted from Albertson and Yelick, 2005) was used in an upper level undergraduate Comparative Vertebrate Biology laboratory classroom at Syracuse University. The first two thirds of the Comparative Vertebrate Biology lab course gave students the opportunity to study the embryology and gross anatomy of several organisms representing various chordate taxa primarily via traditional dissections and the use of models. The final portion of the course involved an innovative approach to teaching anatomy through observation of vertebrate development employing molecular techniques in which WISH was performed on zebrafish embryos. A heterozygous fibroblast growth factor 8 a (fgf8a) mutant line, ace, was used. Due to Mendelian inheritance, ace intercrosses produced wild type, heterozygous, and homozygous ace/fgf8a mutants in a 1:2:1 ratio. RNA probes with known expression patterns in the midline and in developing anatomical structures such as the heart, somites, tailbud, myotome, and brain were used. WISH was performed using zebrafish at the 13 somite and prim-6 stages, with students performing the staining reaction in class. The study of zebrafish embryos at different stages of development gave students the ability to observe how these anatomical structures changed over ontogeny. In addition, some ace/fgf8a mutants displayed improper heart looping, and defects in somite and brain development. The students in this lab observed the normal development of various organ systems using both external anatomy as well as gene expression patterns. They also identified and described embryos displaying improper anatomical development and gene expression (i.e., putative mutants).
For instructors at institutions that do not already own the necessary equipment or where funds for lab and curricular innovation are limited, the financial cost of the reagents and apparatus may be a factor to consider, as will the time and effort required on the part of the instructor regardless of the setting. Nevertheless, we contend that the use of WISH in this type of classroom laboratory setting can provide an important link between developmental genetics and anatomy. As technology advances and the ability to study organismal development at the molecular level becomes easier, cheaper, and increasingly popular, many evolutionary biologists, ecologists, and physiologists are turning to research strategies in the field of molecular biology. Using WISH in a Comparative Vertebrate Biology laboratory classroom is one example of how molecules and anatomy can converge within a single course. This gives upper level college students the opportunity to practice modern biological research techniques, leading to a more diversified education and the promotion of future interdisciplinary scientific research.

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Whole mount in situ hybridization (WISH) was used in an upper level undergraduate Comparative Vertebrate Biology course in addition to vertebrate dissections. This gave students the opportunity to study gene expression patterns as well as gross anatomy, linking the study of molecular and organismal biology within one course.

שימוש הר שלם באתרו הכלאה קישור לביולוגיה מולקולרית האורגניזם

Whole mount in situ hybridization (WISH) is a common technique in molecular biology laboratories used to study gene expression through the localization of ...

Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching morphogenesis. We have developed the limb skin vasculature model to study vascular development in which a pre-existing primitive capillary plexus is reorganized into a hierarchically branched vascular network. Whole-mount confocal microscopy with multiple labelling allows for robust imaging of intact blood vessels as well as their cellular components including endothelial cells, pericytes and smooth muscle cells, using specific fluorescent markers. Advances in this limb skin vasculature model with genetic studies have improved understanding molecular mechanisms of vascular development and patterning. The limb skin vasculature model has been used to study how peripheral nerves provide a spatial template for the differentiation and patterning of arteries. This video article describes a simple and robust protocol to stain intact blood vessels with vascular specific antibodies and fluorescent secondary antibodies, which is applicable for vascularized embryonic organs where we are able to follow the process of vascular development.

We introduce a whole-mount immunohistochemistry and laser scanning confocal microscopy with multiple labelling for analyzing intricate vascular network formation in mouse embryonic limb skin.

Whole-mount עבור ניתוח immunohistochemical vasculature לימב עובריים עור: מערכת מודל לחקר המורפוגנזה מסעף דם בעובר

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching ...

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures are lengthy and technically demanding with multiple important steps that collectively contribute to the quality of the final result. This protocol describes in detail several key quality control steps for optimizing probe labeling and performance. Overall, our protocol provides a detailed description of the critical steps necessary to reproducibly obtain high quality results. First, we describe the generation of digoxygenin (DIG) labeled RNA probes via in vitro transcription of DNA templates generated by PCR. We describe three critical quality control assays to determine the amount, integrity and specific activity of the DIG-labeled probes. These steps are important for generating a probe of sufficient sensitivity to detect endogenous mRNAs in a whole mouse embryo. In addition, we describe methods for the fixation and storage of E8.5-E11.5 day old mouse embryos for in situ hybridization. Then, we describe detailed methods for limited proteinase K digestion of the rehydrated embryos followed by the details of the hybridization conditions, post-hybridization washes and RNase treatment to remove non-specific probe hybridization. An AP-conjugated antibody is used to visualize the labeled probe and reveal the expression pattern of the endogenous transcript. Representative results are shown from successful experiments and typical suboptimal experiments.

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

This whole mount in situ hybridization protocol discusses critical steps that ensure reproducible high quality results for gene expression studies in E8.5-E11.5 day old mouse embryos.

הר שלם<em&gt; באתר</em&gt; הכלאה של E8.5 לעוברי E11.5 עכבר

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures ...

A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract

Development of the lower urogenital tract (LUT) is an intricate process. This complexity is evidenced during formation of the prostate from the fetal male urethra, which relies on androgenic signals and epithelial-mesenchymal interactions1,2. Understanding the molecular mechanisms responsible for prostate development may reveal growth mechanisms that are inappropriately reawakened later in life to give rise to prostate diseases such as benign prostatic hyperplasia and prostate cancer.
The developing LUT is anatomically complex. By the time prostatic budding begins on 16.5 days post conception (dpc), numerous cell types are present. Vasculature, nerves and smooth muscle reside within the mesenchymal stroma3. This stroma surrounds a multilayered epithelium and gives rise to the fetal prostate through androgen receptor-dependent paracrine signals4. The identity of the stromal androgen receptor-responsive genes required for prostate development and the mechanism by which prostate ductal epithelium forms in response to these genes is not fully understood. The ability to precisely identify cell types and localize expression of specific factors within them is imperative to further understand prostate development. In situ hybridization (ISH) allows for localization of mRNAs within a tissue. Thus, this method can be used to identify pattern and timing of expression of signaling molecules and their receptors, thereby elucidating potential prostate developmental regulators.
Here, we describe a high throughput ISH technique to identify mRNA expression patterns in the fetal mouse LUT using vibrating microtome-cut sections. This method offers several advantages over other ISH protocols. Performing ISH on thin sections adhered to a slide is technically difficult; cryosections frequently have poor structural quality while both cryosections and paraffin sections often result in weak signal resolution. Performing ISH on whole mount tissues can result in probe trapping. In contrast, our high throughput technique utilizes thick-cut sections that reveal detailed tissue architecture. Modified microfuge tubes allow easy handling of sections during the ISH procedure. A maximum of 4 mRNA transcripts can be screened from a single 17.5dpc LUT with up to 24 mRNA transcripts detected in a single run, thereby reducing cost and maximizing efficiency. This method allows multiple treatment groups to be processed identically and as a single unit, thereby removing any bias for interpreting data. Most pertinently for prostate researchers, this method provides a spatial and temporal location of low and high abundance mRNA transcripts in the fetal mouse urethra that gives rise to the prostate ductal network.

A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract

Here, we describe an efficient high throughput in situ hybridization (ISH) method for visualizing patterns of mRNA expression in developing fetal mouse prostate tissue sections. The method can be easily adapted to visualize mRNA expression patterns in other mouse tissues or in tissues from other species.

תפוקה גבוהה באתרו הכלאה שיטה לאפיון דפוסי ביטוי mRNA של העכבר עוברית בדרכי השתן ואברי המין התחתון

Development of the lower urogenital tract (LUT) is an intricate process. This complexity is evidenced during formation of the prostate from the fetal male ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...