Name: scanning electron microscopy of macerated tissue to visualize the extracellular matrix
Uploaded: 2016-06-14T15:00:00.000+00:00
Duration: 10 min 21 s
Description: Watch this Scientific Journal Video about Scanning Electron Microscopy of Macerated Tissue to Visualize the Extracellular Matrix at JoVE.com

This study was funded by grants from the National Institutes of Health (NIH), Heart, Lung, and Blood Institute (NIHLB): K01-HL-121045, K08-HL-094703, 5T32HL007411-35, P20 HL101425, U01 HL100398. 
Imaging and tissue processing (after NaOH maceration) were performed through the use of the Vanderbilt University Medical Center (VUMC) Cell Imaging Shared Resource (CISR) (supported by NIH grants CA68485, DK20593, DK58404, DK59637 and EY08126). We are especially grateful to the VUMC CISR core directors (Dr. Sam Wells and Dr. W. Gray (Jay) Jerome) for valuable technical advice and also for providing core space and resources for the purposes of filming the technique highlighted in this paper. 
We would like to extend our deepest appreciation to Dr. Yan Ru Su and Ms. Kelsey Tomasek in the Cardiology Core Lab for Translational and Clinical Research at Vanderbilt University for providing technical expertise and for collecting human tissue samples used in this study.

משפט ואתיקה: הפרוטוקולים עבור טיפול בבעלי חיים אושרו על ידי הטיפול בבעלי החיים המוסדי ונדרבילט ועדת שימוש (IACUC, פרוטוקולי מספר M / 10/117 (חזירים) ו- M / 10/219 (עכברים) ו נערכים על פי תקני AAALAC-בינלאומיים. שימוש של רקמות לב אנושיות פקידה אושר על ידי IRB במרכז הרפואי של האוניברסיטה ונדרבילט (מספר פרוטוקול 100,887). לדוגמא 1. איסוף ואחסון <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הפוך glutaraldehyde 4% טרי ב 0.1 M חיץ פוספט פתרון (PB). זהירות: Glutaraldehyde הוא רעיל, ללבוש כפפות ולעבוד במנדף. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> בצע פתרון 0.2 M המניות של PB, pH 7.4 באמצעות 21.8 גרם Na 2 HPO 4 ו -6.4 גרם לאא 2 4 PO. Quantum satis (QS) 1,000 מ&quot;ל DH 2 O. </li><li style=";text-align:right;direction:rtl"> להוסיף 400 μl של glutaraldehyde 50% עד 2.5 מ&quot;ל של פתרון המניות PB ו -2.1 מ&quot;ל DH 2 O. </li></ol></li><li style=";text-align:right;direction:rtl"> לטבול נתח (לא גדול מ -2 גמ &#39;2) של רקמה לתוך פתרון glutaraldehyde 4%. הערה: חתיכות קטנות יכולות לשמש גם אבל צריך להיות בגודל מספיק (לא קטן מ -5 מ&quot;מ 2) להיות מדמיינים בקלות על ידי עין על מנת להקל צעדים מאוחר יותר של הפרוטוקול. </li><li style=";text-align:right;direction:rtl"> דגירה בטמפרטורת החדר למשך שעה 1, ולאחר מכן לאחסן לזמן בלתי מוגבל ב 4 ° C.. </li></ol> 2. Decellularization של רקמת לב <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הפוך בתמיסה מימית NaOH 10% טריים באמצעות 10 כדורי גרם NaOH / 100 מ&quot;ל DH 2 O. זהירות: פתרונות NaOH הם מאכלים והוא יכול לגרום אלקלי כוויות, ללבוש כפפות. </li><li style=";text-align:right;direction:rtl"> לשטוף רקמות ב DH 2 O. </li><li style=";text-align:right;direction:rtl"> דגירה בתמיסה 10% NaOH בטמפרטורת החדר למשך 6 - 10 ימים (עד שינויים ברקמה מן אדמדם-חום מחוץ לבן או לבן). </li><li style=";text-align:right;direction:rtl"> יש לשטוף ב DH 2 O עד הרקמה הופכת שקופה. </li><li style=";text-align:right;direction:rtl"> לטבול רקמות בחומצה טאני 1% ל -4 שעות בטמפרטורת החדר. השתמש 1 מ&quot;ל 5% מניות פתרון לכל 4 מ&quot;ל DH 2 O. זהירות: חומצה טאניתהוא מגרה חזק, ללבוש כפפות. </li><li style=";text-align:right;direction:rtl"> יש לשטוף ב DH 2 O לילה. </li></ol> 3. Osmication והתייבשות של רקמת לב (ב פומה הוד לבטיחות) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> בצע פתרון 0.2 M המניות של חיץ נתרן cacodylate באמצעות cacodylate 21.4 גרם נתרן, סידן כלורי 10.0 גרם ו 450 מ&quot;ל DH 2 O. מערבבים, ולאחר מכן להוסיף חומצה הידרוכלורית לפי הצורך כדי להתאים את pH 7.4. Qs 500 מ&quot;ל עם DH 2 O. זהירות: cacodylate נתרן וחומצה הידרוכלורית רעילים, ללבוש כפפות ולעבוד במנדף. </li><li style=";text-align:right;direction:rtl"> הפוך פתרון המניות 2% מימית של tetroxide אוסמיום במנדף לבטיחות ידי המסת גביש tetroxide אוסמיום 1 גרם ב 50 מ&quot;ל DH 2 O. זהירות: tetroxide אוסמיום הוא מפגע משאיפת חמור; קיבעון האדים של ממברנות ריר או העיניים אפשרי, ומכאן להתמודד רק במנדף עם כפפות. שומר סנסציה מומלצת. </li><li style=";text-align:right;direction:rtl"> לשטוף רקמות במאגר cacodylate 0.1 M נתרן (לערבב פתרון מניות 1: 1 עם DH <sub&gt; 2 O) במשך 5 דקות על הכתף (או תסיסה עדינה). </li><li style=";text-align:right;direction:rtl"> חזור על השלב הקודם פעמיים, עבור סכום כולל של שלוש שטיפות חיץ. </li><li style=";text-align:right;direction:rtl"> לטבול רקמות tetroxide אוסמיום 1% ב 0.1 M נתרן חיץ cacodylate (לערבב cacodylate נתרן המניות אוסמיום המניות tetroxide 1: 1) על הכתף במשך שעה 1. </li><li style=";text-align:right;direction:rtl"> לשטוף רקמות 0.1 M נתרן cacodylate חיץ 3 פעמים במשך 5 דקות כל אחד על הכתף. </li><li style=";text-align:right;direction:rtl"> לשטוף רקמות באמצעות ריכוזים גוברים של אתנול (30%, 50%, 75%, 85%, 95%, ולבסוף 100%) במשך 15 דקות כל אחד על כתף. </li></ol> 4. הכנת פני שטח חתך עבור SEM <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> העברת רקמות באתנול 100% כדי בצלחת פטרי רדודה גם המכילה אתנול 100%. </li><li style=";text-align:right;direction:rtl"> להחזיק שני סכיני גילוח חד מאוד כך ולדירות הצדדים נמצאות בקשר אחד עם השני, וצלב קצות החיתוך ליצירת שני צדדים של משולש שווה צלעות מעל הדגימה. כדי להשיג זאת, להשתמש ביד שמאל למקום להב אחד בצד הימני או השמאלי של המדגםחתך שמאלה. במקביל, השתמש ביד ימין להציב את הלהב השני בשמאל הקיצוני של ימינה המדגם פרוס. לפיכך, הלהבים יהיו החליקו אחד נגד השני מכיוונים מנוגדים לעשות חתך אחד חלקה. </li><li style=";text-align:right;direction:rtl"> חלק את צדדי להב שטוח אחד נגד השני כדי לעשות חתך מאוד נקי של הדגימה עם עיוות מינימאלית או לקרוע כוח, רצוי לחשוף שטח פנים גדול ככל האפשר מבלי לפגוע הדגימה. </li><li style=";text-align:right;direction:rtl"> חזור על פעולה עבור כל דגימה לחשוף משטחי החתך האפשריים הנקיים לבדיקה ב SEM. </li></ol> 5. ייבוש נקודה קריטית (CPD) של רקמת לב <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> השתמש מרית או פינצטה להעביר רקמות לבעל המדגם של מייבש הנקודה הקריטי (CPD), להבטיח כי נשארה רקמה באתנול 100% בכל העת. ודא כי בעל שקוע אתנול כי העברת מושגת עם הרקמה החשופה לאוויר לא יותר מכמה שניות. </li><li style=";text-align:right;direction:rtl"> Operate CPD לכל לנוהוראות אה להשלים 3 טיהור-ו-למלא מחזורים להחליף אתנול עם פחמן דו חמצני נוזלי. </li><li style=";text-align:right;direction:rtl"> Operate CPD לכל ידנית של המשתמש כדי להשיג ייבוש נקודה קריטית של דגימות ולהחזיר אותם בלחץ אטמוספרי עם אוורור מבוקר של CO 2. </li></ol> שמת 6. דוגמאות רקמה לב עבור SEM <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> כן בדל מדגם SEM עבור כל דגימה, על ידי דבקות כרטיסייה דבקה פחמן אל פני השטח העליון של בדל האלומיניום. </li><li style=";text-align:right;direction:rtl"> בעזרת סטראו, בזהירות לדבוק דגימה ללשונית הדבקה עם משטח החתך של עניין פונה כלפי מעלה (הרחק הכרטיסייה, גלויה), וקרוב ככל האפשר מקבילה עם המטוס של פני שטח בדל מדגם. אין לחקור או לגעת במשטח של עניין. </li><li style=";text-align:right;direction:rtl"> לשבור מקל מוליך עץ כדי להשיג מברשת מחודדת, אידיאלית עבור יישום צבע. החל כסף או צבע פחמן בבסיס והדפנות של הדגימה, כדי לשפר את דבקות הבדל. </li><li style=";text-align:right;direction:rtl"> הרחב קו דק מאוד של כסף או פחמן לצייר עד יתרון של פני השטח של עניין, כדי לספק נתיב תשלום מפני השטח של עניין הקרקע. </li><li style=";text-align:right;direction:rtl"> החל 2 או 3 מורח קטן של כסף או צבע פחמן מסביב בכרטיסיית פחמן, כדי לספק נתיב מוליך מפני שטח כרטיסיית פחמן אל בדל מתכת ובכך לקרקע. </li><li style=";text-align:right;direction:rtl"> אפשר צבע מוליך להתייבש במשך שתי שעות. </li><li style=";text-align:right;direction:rtl"> Operate coater גמגום לכל מדריך למשתמש להחיל ציפוי כבד יחסית (טווח היעד של 30 - 40 ננומטר) של סגסוגת זהב-פלדיום או זהב. משאבת תא דגימה כ -0.1 mbar; כוונו את הטיימר ל -40 שניות. שסתום סט פתוח 08:00 המיקום (זרימת גז ארגון מתון). לחצו על התחל כדי ליזום ציפוי גמגום 30 מילי-אמפר. הפרשות זוהרות סגולות צריכות להיות גלויות בתא הדגימה. </li></ol> 7. בדיקת SEM של דגימות רקמת לב <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לבצע סריקה מיקרוסקופית אלקטרונים במתח האצה נמוך יחסית כדי למזער הדמיה problems הקשורים פיזור חסותה המסכן במדגם (טעינה). מצב הדמיה הראשונית מוצעים הם: 5 kV מאיץ מתח, 10 מ&quot;מ ומרחק עבודה. </li><li style=";text-align:right;direction:rtl"> עם סיוע של מפעיל מנוסה, להעסיק גדל עובד מרחק להאריך עומק השדה בתנאי הדמיה הדורשים מוקד הסיבים במטוסים מספר מוקדים, או סיבי הארכה עבור אורך ניכר בממד z. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> עבור המיקרוסקופ משמש כאן (ראה טבלה של חומרים), בתוך גישת הממשק המשתמש על כרטיסיית הניווט ב ימני עליון. </li><li style=";text-align:right;direction:rtl"> גש לכרטיסיית הקואורדינטות מתפריט השלב. כדי להגדיל מרחק עבודה, זן ערך גדול במילימטרים עבור Z קואורדינטות ואז ללחוץ על עבור לכרטיסייה כדי להזיז את הבמה מדגם למרחק עובד נכנס. </li></ol></li><li style=";text-align:right;direction:rtl"> עם סיוע של מפעיל מנוסה, להעסיק הטית דגימה וסיבוב כדי למקם את השטח של מאונך המעניין את אלומת האלקטרונים. הטיה נוספת של 10 עד 30 degrEES מתוך עמדה זו עשויה לשפר תצפית ותיעוד של המבנה מטריקס. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> עבור המיקרוסקופ משמש כאן (ראה טבלה של חומרים), בממשק המשתמש, לחץ לחיצה ממושכת על כפתור העכבר הימני, ולאחר מכן חלק שמאלה או ימינה כדי למקד את הדגימה ליד בפריפריה של מטוס המשטח המוכן של עניין, תוך ציון מרחק העבודה הממוקד . </li><li style=";text-align:right;direction:rtl"> נווט עם ג&#39;ויסטיק הממשק הידני על מנת לעבור ליד הקצה היריב של מוקד השטח וחזור. אם עובדי מרחק אינו שווה בערך במיקום הראשון, דגימה להטות להשיג סכם משוער בשני המקומות באמצעות לשונית הקואורדינטות של תפריט השלב (ראה 7.2) והזנת ערך הטיה של T לתאם. </li><li style=";text-align:right;direction:rtl"> סובב דגימה תשעים מעלות (להזין ערך לתוך R לתאם שדה) וחזור על התהליך עד שכל העמדות ממוקדות על מרחק העבודה הזהה כ. הערה: SEM לחץ משתנה עשוי להיות מועסק על מנת לשפר את פיזור תשלוםאם זמין. ואקום גבוה הוא מצב הפעלה סטנדרטית עבור במיקרוסקופ אלקטרונים סורק. </li></ol></li></ol>

The authors have nothing to disclose.

הכנת שטח חתך היא השלב הקריטי ביותר במהלך הפרוטוקול. כדי לשמר מבנה עדין, דגימות מיובשות חייבות להישאר באתנול 100% בכל העת עד הציגו את תהליך ייבוש נקודה הקריטי. לכן החיתוך של דגימות כדי להשיג משטחים לבדיקה EM חייב להיעשות תוך דגימות קומטי באתנול בצלחת רדודה. זה גם קריטי, כי...

Fibrosis disrupts the normal myocardial collagen network, which is critical for normal mechanistic functions of cardiomyocytes 1,2 as well as for inter-cellular communication, intracellular signaling, and cell survival 3. The development of fibrosis is a major determinant of cardiac function, and fibrotic remodeling of the cardiac interstitium arising from a variety of etiologies leads to increased left ventricular stiffness and diastolic dysfunction 4. Myocardial fibrosis may also lead to arrhythmias, and whether the progression of fibrotic remodeling is a general or local phenomenon, it is highly associated with a poor prognosis in patients with ischemic and non-ischemic cardiomyopathy 5. Likewise, the absence of myocardial fibrosis is a strong predictor of ventricular functional recovery and long-term survival 6. 
The hallmark of fibrosis is the deposition of excess collagen, which has the tensile strength of steel 7 and can adversely affect cardiomyocyte function on multiple levels. Mechanical forces resulting from an excessively collagenous matrix can lead to cardiomyocyte atrophy 8,9, passive tissue stiffness 10, tonic contraction-induced myocardial stiffness 11-13, and reduced delivery of oxygen to the remaining population of cardiomyocytes. Gap junction coupling of cardiomyocytes and myoFbs can also compromise the heart's electrical characteristics, creating a greater risk for the development of arrhythmias 14-16. Perivascular fibrosis alters vasomotor reactivity of intramural coronary arteries and arterioles 17 and contributes to luminal narrowing that reduces the supply of oxygen and thus the survival of cardiomyocytes 17-22. Pathogenic fibrotic and electrical remodeling, emanating from an initial site of ischemic injury or energy imbalance, inevitably progresses to heart failure. 
Cardiomyocyte necrosis initiates the fibrotic response, and subsequent adverse fibrotic remodeling can occur irrespective of etiology. Finding a way to control cardiac fibrosis would be clinically beneficial for the treatment of ischemic and idiopathic cardiomyopathies, hypertensive heart disease, hypertrophic cardiomyopathy, valvular heart disease and dystrophinopathies 23-42. Regardless of how the fibrotic disease process begins, soluble, profibrotic factors can cross the interstitial space and provoke activation of interstitial and adventitial fibroblasts at sites remote to the initial fibrotic scar, creating a cascade effect that ultimately leads to heart failure. The optimum scenario would be to exploit the fibrillogenic process using a targeted therapeutic that can be applied during the compensative hypertrophic stage of cardiomyopathy before it progresses to systolic pump failure, diastolic heart failure, or other end-stage outcomes. The ultimate goal would be to reverse fibrosis so that dead cardiomyocytes can be replaced and heart function restored completely. 
The importance of the matrix is widely understood, yet methods to study the matrix are limited mainly to quantitative measurements of major structural components, particularly collagen, and relative levels of different matrix and matricellular proteins. This protocol highlights a rarely used technique that is useful for assessing qualitative differences in the cardiac matrix. This technique has been recently used to compare and contrast fundamental differences in heart matrices from different etiologies of heart disease (in human explants), to examine hearts from post-infarcted pigs treated with the glial growth factor (GGF) isoform of neuregulin-1&#946;, relative to untreated animals 43, and to probe for differences in the matrices of cardiac tissues from mdx mice (a commonly used animal model of Duchenne Muscular Dystrophy) at different ages and compared to wild-type controls. This technique was first introduced by Drs. Caulfield and Borg in 1979 44, but few studies have since employed this powerful technique 45-47, re-introduced here with only slight modification. This methodology is a valuable research tool, because it provides qualitative information about extracellular matrix ultrastructure that might otherwise be overlooked when simply measuring matrix component content and/or level of fibrosis.

<table><tbody><tr><td>Calcium Chloride</td><td>Electron Microscopy Sciences</td><td>12340</td><td>100 g</td></tr><tr><td>Carbon Adhesive</td><td>Electron Microscopy Sciences</td><td>12664</td><td>30 g</td></tr><tr><td>Carbon Adhesive Tabs</td><td>Electron Microscopy Sciences</td><td>77825</td><td>order to fit stubs</td></tr><tr><td>Double Edge Razor Blades Stainless Steel</td><td>Ted Pella, Inc</td><td>121-6</td><td>250/pkg</td></tr><tr><td>Ethanol</td><td>Electron Microscopy Sciences</td><td>15055</td><td>450 ml</td></tr><tr><td>Gluteraldehyde, 50% Solution</td><td>Electron Microscopy Sciences</td><td>16310</td><td>EM grade, distillation purified</td></tr><tr><td>Hydrochloric Acid</td><td>Electron Microscopy Sciences</td><td>16760 or 16770</td><td>100 ml</td></tr><tr><td>Monosodium Phosphate NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>S9251-250G</td><td>250 g</td></tr><tr><td>Osmium Tetroxide</td><td>Electron Microscopy Sciences</td><td>19100</td><td>1 g</td></tr><tr><td>Silver Conductive Adhesive</td><td>Electron Microscopy Sciences</td><td>12686-15</td><td>15 g</td></tr><tr><td>Sodium hydroxide (NaOH)</td><td>Sigma-Aldrich</td><td>S8045-1KG</td><td>1 kg</td></tr><tr><td>Sodium Phosphate Dibasic (Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>S3264-500G</td><td>500 g</td></tr><tr><td>Tannic Acid, 5% Aqueous&amp;nbsp;</td><td>Electron Microscopy Sciences</td><td>21702-5</td><td>500 ml</td></tr><tr><td>Trihydrate Sodium Cacodylate</td><td>Electron Microscopy Sciences</td><td>12300</td><td>100 g</td></tr><tr><td>Gold-palladium Alloy or Gold</td><td>Refining Systems, Inc.&amp;nbsp;</td><td>varies</td><td>specific to the sputter coater make and model</td></tr><tr><td>Critical Point Dryer</td><td>Electron Microscopy Sciences</td><td>850</td><td></td></tr><tr><td>Plain Wooden Applicators</td><td>Fisher Scientific</td><td>23-400-102</td><td></td></tr><tr><td>Quanta 250 Environmental SEM</td><td>FEI</td><td>Q250 SEM</td><td></td></tr><tr><td>Sputter Coater</td><td>Cressington Scientific Instruments Ltd.</td><td>Model 108</td><td></td></tr><tr><td>Alluminum SEM Sample Stubs</td><td>Electron Microscopy Sciences</td><td>75220-12</td><td>specific to the miscroscope</td></tr></tbody></table>

scanning electron microscopy of macerated tissue to visualize the extracellular matrix

Fibrosis is a component of all forms of heart disease regardless of etiology, and while much progress has been made in the field of cardiac matrix biology, there are still major gaps related to how the matrix is formed, how physiological and pathological remodeling differ, and most importantly how matrix dynamics might be manipulated to promote healing and inhibit fibrosis. There is currently no treatment option for controlling, preventing, or reversing cardiac fibrosis. Part of the reason is likely the sheer complexity of cardiac scar formation, such as occurs after myocardial infarction to immediately replace dead or dying cardiomyocytes. The extracellular matrix itself participates in remodeling by activating resident cells and also by helping to guide infiltrating cells to the defunct lesion. The matrix is also a storage locker of sorts for matricellular proteins that are crucial to normal matrix turnover, as well as fibrotic signaling. The matrix has additionally been demonstrated to play an electromechanical role in cardiac tissue. Most techniques for assessing fibrosis are not qualitative in nature, but rather provide quantitative results that are useful for comparing two groups but that do not provide information related to the underlying matrix structure. Highlighted here is a technique for visualizing cardiac matrix ultrastructure. Scanning electron microscopy of decellularized heart tissue reveals striking differences in structure that might otherwise be missed using traditional quantitative research methods.

הטכניקה המודגשת יושמה לרקמות לב מתורם השתלת לב אנושית בשימוש (איור 1), רקמות explanted מן המושתלים, לבבות מ wild-type ועכברים דיסטרופי (איור 3), ו בדגימות לב אוטם שלאחר שריר לב מתוך חזירים מודל של פגיעה לבבית (איור 2). כפי שניתן לראות <st...

Watch this Scientific Journal Video about Scanning Electron Microscopy of Macerated Tissue to Visualize the Extracellular Matrix at JoVE.com

Scanning Electron Microscopy of Macerated Tissue to Visualize the Extracellular Matrix

Shown here is a method for visualizing extracellular matrix ultrastructure in decellularized cardiac tissues.

מיקרוסקופ אלקטרוני סורק של רקמות macerated לדמיין את תאי מטריקס

Fibrosis is a component of all forms of heart disease regardless of etiology, and while much progress has been made in the field of cardiac matrix ...

scanning-electron-microscopy-macerated-tissue-to-visualize

Research

JoVE Journal

Biology

9.9K Views.  Vanderbilt University Medical Center. Shown here is a method for visualizing extracellular matrix ultrastructure in decellularized cardiac tissues.

Video: Scanning Electron Microscopy of Macerated Tissue to Visualize the Extracellular Matrix

Glycoproteomics of the Extracellular Matrix: A Method for Intact Glycopeptide Analysis Using Mass Spectrometry

Fibrosis is a hallmark of many cardiovascular diseases and is associated with the exacerbated secretion and deposition of the extracellular matrix (ECM). Using proteomics, we have previously identified more than 150 ECM and ECM-associated proteins in cardiovascular tissues. Notably, many ECM proteins are glycosylated. This post-translational modification affects protein folding, solubility, binding, and degradation. We have developed a sequential extraction and enrichment method for ECM proteins that is compatible with the subsequent liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of intact glycopeptides. The strategy is based on sequential incubations with NaCl, SDS for tissue decellularization, and guanidine hydrochloride for the solubilization of ECM proteins. Recent advances in LC-MS/MS include fragmentation methods, such as combinations of higher-energy collision dissociation (HCD) and electron transfer dissociation (ETD), which allow for the direct compositional analysis of glycopeptides of ECM proteins. In the present paper, we describe a method to prepare the ECM from tissue samples. The method not only allows for protein profiling but also the assessment and characterization of glycosylation by MS analysis.

This paper describes a methodology to prepare cardiovascular tissue samples for MS analysis that allows for (1) the analysis of ECM protein composition, (2) the identification of glycosylation sites, and (3) the compositional characterization of glycan forms. This methodology can be applied, with minor modifications, to the study of the ECM in other tissues.

Glycoproteomics של מטריקס: שיטה Intact ניתוח Glycopeptide באמצעות ספקטרומטריית מסה

Fibrosis is a hallmark of many cardiovascular diseases and is associated with the exacerbated secretion and deposition of the extracellular matrix (ECM). ...

Biochemistry

Observing and Quantifying Fibroblast-mediated Fibrin Gel Compaction

Cells embedded in collagen and fibrin gels attach and exert traction forces on the fibers of the gel. These forces can lead to local and global reorganization and realignment of the gel microstructure. This process proceeds in a complex manner that is dependent in part on the interplay between the location of the cells, the geometry of the gel, and the mechanical constraints on the gel. To better understand how these variables produce global fiber alignment patterns, we use time-lapse differential interference contrast (DIC) microscopy coupled with an environmentally controlled bioreactor to observe the compaction process between geometrically spaced explants (clusters of fibroblasts). The images are then analyzed with a custom image processing algorithm to obtain maps of the strain. The information obtained from this technique can be used to probe the mechanobiology of various cell-matrix interactions, which has important implications for understanding processes in wound healing, disease development, and tissue engineering applications.

Time-lapse microscopy and image processing techniques were used to observe and analyze fibroblast-mediated gel compaction and fibrin fiber realignment in an environmentally controlled bioreactor over a 48 hr&#160;period.

התבוננות וכימות דחיסה של ג'ל פיברובלסט בתיווך פיברין

Cells embedded in collagen and fibrin gels attach and exert traction forces on the fibers of the gel. These forces can lead to local and global ...

Bioengineering

Long-term Intravital Immunofluorescence Imaging of Tissue Matrix Components with Epifluorescence and Two-photon Microscopy

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue function during development and organ homeostasis. It does so by acting via biochemical, biomechanical, and biophysical signaling pathways, such as through the release of bioactive ECM protein fragments, regulating tissue tension, and providing pathways for cell migration. The extracellular matrix of the tumor microenvironment undergoes substantial remodeling, characterized by the degradation, deposition and organization of fibrillar and non-fibrillar matrix proteins. Stromal stiffening of the tumor microenvironment can promote tumor growth and invasion, and cause remodeling of blood and lymphatic vessels. Live imaging of matrix proteins, however, to this point is limited to fibrillar collagens that can be detected by second harmonic generation using multi-photon microscopy, leaving the majority of matrix components largely invisible. Here we describe procedures for tumor inoculation in the thin dorsal ear skin, immunolabeling of extracellular matrix proteins and intravital imaging of the exposed tissue in live mice using epifluorescence and two-photon microscopy. Our intravital imaging method allows for the direct detection of both fibrillar and non-fibrillar matrix proteins in the context of a growing dermal tumor. We show examples of vessel remodeling caused by local matrix contraction. We also found that fibrillar matrix of the tumor detected with the second harmonic generation is spatially distinct from newly deposited matrix components such as tenascin C. We also showed long-term (12 hours) imaging of T-cell interaction with tumor cells and tumor cells migration along the collagen IV of basement membrane. Taken together, this method uniquely allows for the simultaneous detection of tumor cells, their physical microenvironment and the endogenous tissue immune response over time, which may provide important insights into the mechanisms underlying tumor progression and ultimate success or resistance to therapy.

The extracellular matrix undergoes substantial remodeling during wound healing, inflammation and tumorigenesis. We present a novel intravital immunofluorescence microscopy approach to visualize the dynamics of fibrillar as well as mesh-like matrix components with high spatial and temporal resolution using epifluorescence or two-photon microscopy.

לטווח ארוך Intravital Immunofluorescence הדמיה של רקמות מטריקס רכיבים עם Epifluorescence ומיקרוסקופי שני פוטונים

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue ...

Revealing the Cytoskeletal Organization of Invasive Cancer Cells in 3D

Cell migration has traditionally been studied in 2D substrates. However, it has become increasingly evident that there is a need to study cell migration in more appropriate 3D environments, which better resemble the dimensionality of the physiological processes in question. Migratory cells can substantially differ in their morphology and mode of migration depending on whether they are moving on 2D or 3D substrates. Due to technical difficulties and incompatibilities with most standard protocols, structural and functional analysis of cells embedded within 3D matrices still remains uncommon. This article describes methods for preparation and imaging of 3D cancer cell cultures, either as single cells or spheroids. As an appropriate ECM substrate for cancer cell migration, we use nonpepsinized rat tail collagen I polymerized at room-temperature and fluorescently labeled to facilitate visualization using standard confocal microscopes. This work also includes a protocol for 3D immunofluorescent labeling of endogenous cell cytoskeleton. Using these protocols we hope to contribute to a better description of the molecular composition, localization, and functions of cellular structures in 3D.

This article presents a method to fluorescently label collagen that can be further used for both fix and live imaging of 3D cell cultures. We also provide an optimized protocol to visualize endogenous cytoskeletal proteins of cells cultured in 3D environments.

חושף את ארגון cytoskeletal של תאי סרטן פולשני ב-3D

Cell migration has traditionally been studied in 2D substrates. However, it has become increasingly evident that there is a need to study cell migration ...

Medicine

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern tissue engineering. A technical bottleneck is the deposition of collagen in vitro, as it is notoriously slow, resulting in sub-optimal formation of connective tissue and subsequent tissue cohesion, particularly in skin models. Here, we describe a method which involves the addition of differentially-sized sucrose co-polymers to skin cultures to generate macromolecular crowding (MMC), which results in a dramatic enhancement of collagen deposition. Particularly, dermal fibroblasts deposited a significant amount of collagen I/IV/VII and fibronectin under MMC in comparison to controls.
The protocol also describes a method to decellularize crowded cell layers, exposing significant amounts of extracellular matrix (ECM) which were retained on the culture surface as evidenced by immunocytochemistry. Total matrix mass and distribution pattern was studied using interference reflection microscopy. Interestingly, fibroblasts, keratinocytes and co-cultures produced cell-derived matrices (CDM) of varying composition and morphology. CDM could be used as &#34;bio-scaffolds&#34; for secondary cell seeding, where the current use of coatings or scaffolds, typically from xenogenic animal sources, can be avoided, thus moving towards more clinically relevant applications.
In addition, this protocol describes the application of MMC during the submerged phase of a 3D-organotypic skin co-culture model which was sufficient to enhance ECM deposition in the dermo-epidermal junction (DEJ), in particular, collagen VII, the major component of anchoring fibrils. Electron microscopy confirmed the presence of anchoring fibrils in cultures developed with MMC, as compared to controls. This is significant as anchoring fibrils tether the dermis to the epidermis, hence, having a pre-formed mature DEJ may benefit skin graft recipients in terms of graft stability and overall wound healing. Furthermore, culture time was condensed from 5 weeks to 3 weeks to obtain a mature construct, when using MMC, reducing costs.

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

We present a protocol to obtain cell-derived matrices rich in extracellular matrix proteins, using macromolecular crowders (MMC). In addition, we present a protocol which incorporates MMC in 3D organotypic skin co-culture generation, which reduces culture time while maintaining maturity of construct.

עור 2D and 3D שיפור<em&gt; במבחנה</em&gt; מודלים שימוש להצטופפות מולקולארית

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern ...

Visualizing Macrophage Extracellular Traps Using Confocal Microscopy

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal microscopy methods to visualize macrophage extracellular traps (METs). These extracellular traps are defined by the presence of extracellular chromatin with co-expression of other components such as granule proteases, citrullinated histones, and peptidyl arginase deiminase (PAD). The expression of METs is generally measured after exposure to a stimulus and compared to un-stimulated samples. Samples are also included for background and isotype control. Cells are analyzed using well-defined image analysis software. Confocal microscopy may be used to define the presence of METs both in vitro and in vivo in lung tissue.

Macrophage extracellular traps are a newly described entity. This article will concentrate on confocal microscopy methods and how they are visualized in vitro and in vivo from lung samples.

להמחיש מלכודות חוץ-תאי מקרופאג באמצעות מיקרוסקופיה קונפוקלית

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal ...

Immunology and Infection

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts 

Cellular invasion into local tissues is a process important in development and homeostasis. Malregulated invasion and subsequent cell movement is characteristic of multiple pathological processes, including inflammation, cardiovascular disease and tumor cell metastasis1. Focalized proteolytic degradation of extracellular matrix (ECM) components in the epithelial or endothelial basement membrane is a critical step in initiating cellular invasion. In tumor cells, extensive in vitro analysis has determined that ECM degradation is accomplished by ventral actin-rich membrane protrusive structures termed invadopodia2,3. Invadopodia form in close apposition to the ECM, where they moderate ECM breakdown through the action of matrix metalloproteinases (MMPs). The ability of tumor cells to form invadopodia directly correlates with the ability to invade into local stroma and associated vascular components3. 
Visualization of invadopodia-mediated ECM degradation of cells by fluorescent microscopy using dye-labeled matrix proteins coated onto glass coverslips has emerged as the most prevalent technique for evaluating the degree of matrix proteolysis and cellular invasive potential4,5. Here we describe a version of the standard method for generating fluorescently-labeled glass coverslips utilizing a commercially available Oregon Green-488 gelatin conjugate. This method is easily scaled to rapidly produce large numbers of coated coverslips. We show some of the common microscopic artifacts that are often encountered during this procedure and how these can be avoided. Finally, we describe standardized methods using readily available computer software to allow quantification of labeled gelatin matrix degradation mediated by individual cells and by entire cellular populations. The described procedures provide the ability to accurately and reproducibly monitor invadopodia activity, and can also serve as a platform for evaluating the efficacy of modulating protein expression or testing of anti-invasive compounds on extracellular matrix degradation in single and multicellular settings.

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts

We describe the prototypical method for producing microscope coverslips coated with fluorescent gelatin for visualizing invadopodia-mediated matrix degradation. Computational techniques using available software are presented for quantifying the resultant levels of matrix proteolysis by single cells within a mixed population and for multicellular groups encompassing entire microscopic fields.

מדידת כמותית של Proteolysis מטריקס invadopodia בתיווך בקשרים יחידים ורבים תאיים

Cellular invasion into local tissues is a process important in development and homeostasis.  Malregulated invasion and subsequent cell movement is ...

Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of cell proliferation, survival, migration, etc. The ECM plays important roles in development and in diverse pathologies including cardio-vascular and musculo-skeletal diseases, fibrosis, and cancer. Thus, characterizing the composition of ECMs of normal and diseased tissues could lead to the identification of novel prognostic and diagnostic biomarkers and potential novel therapeutic targets. However, the very nature of ECM proteins (large in size, cross-linked and covalently bound, heavily glycosylated) has rendered biochemical analyses of ECMs challenging. To overcome this challenge, we developed a method to enrich ECMs from fresh or frozen tissues and tumors that takes advantage of the insolubility of ECM proteins. We describe here in detail the decellularization procedure that consists of sequential incubations in buffers of different pH and salt and detergent concentrations and that results in 1) the extraction of intracellular (cytosolic, nuclear, membrane and cytoskeletal) proteins and 2) the enrichment of ECM proteins. We then describe how to deglycosylate and digest ECM-enriched protein preparations into peptides for subsequent analysis by mass spectrometry.

This protocol describes a procedure for enriching ECM proteins from tissues or tumors and deglycosylating and digesting the ECM-enriched preparations into peptides to analyze their protein composition by mass spectrometry.

העשרה תאית מטריקס חלבונים מרקמות ועיכול לפפטידים לניתוח ספקטרומטריית המסה

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of ...

A Rapid, Scalable Method for the Isolation, Functional Study, and Analysis of Cell-derived Extracellular Matrix

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and pathological processes. However, the isolation of ECM, from tissues or cell culture, is complicated by the insoluble and cross-linked nature of the assembled ECM and by the potential contamination of ECM extracts with cell surface and intracellular proteins. Here, we describe a method for use with cultured cells that is rapid and reliably removes cells to isolate a cell-derived ECM for downstream experimentation.
Through use of this method, the isolated ECM and its components can be visualized by in situ immunofluorescence microscopy. The dynamics of specific ECM proteins can be tracked by tracing the deposition of a tagged protein using fluorescence microscopy, both before and after the removal of cells. Alternatively, the isolated ECM can be extracted for biochemical analysis, such as sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. At larger scales, a full proteomics analysis of the isolated ECM by mass spectrometry can be conducted. By conducting ECM isolation under sterile conditions, sterile ECM layers can be obtained for functional or phenotypic studies with any cell of interest. The method can be applied to any adherent cell type, is relatively easy to perform, and can be linked to a wide repertoire of experimental designs.

The extracellular matrix plays a major role in defining the microenvironment of cells and in modulating cell behavior and phenotype. We describe a rapid method for the isolation of cell-derived extracellular matrix, which can be adapted to different scales for microscopic, biochemical, proteomic, or functional studies.

מהיר, שיטת Scalable עבור הבידוד, הלימוד פונקציונלי, וניתוח של מטריקס תאים שמקורם

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and ...

Scanning Electron Microscopy (SEM)

Source: Laboratory of Dr. Andrew J. Steckl &#8212; University of Cincinnati
A scanning electron microscope, or SEM, is a powerful microscope that uses electrons to form an image. It allows for imaging of conductive samples at magnifications that cannot be achieved using traditional microscopes. Modern light microscopes can achieve a magnification of ~1,000X, while typical SEM can reach magnifications of more than 30,000X.&#160;Because the SEM doesn&#8217;t use light to create images, the resulting pictures it forms are in black and white.&#160;
Conductive samples are loaded onto the SEM&#8217;s sample stage. Once the sample chamber reaches vacuum, the user will proceed to align the electron gun in the system to the proper location. The electron gun shoots out a beam of high-energy electrons, which travel through a combination of lenses and apertures and eventually hit the sample. As the electron gun continues to shoot electrons at a precise position on the sample, secondary electrons will bounce off of the sample. These secondary electrons are identified by the detector. The signal found from the secondary electrons is amplified and sent to the monitor, creating a 3D image. This video will demonstrate SEM sample preparation, operation, and imaging capabilities.

Scanning Electron Microscopy SEM: Operation, Image Analysis

סריקת מיקרוסקופיית אלקטרונים (SEM)

Source: Laboratory of Dr. Andrew J. Steckl &#8212; University of Cincinnati
A scanning electron microscope, or SEM, is a powerful microscope that uses ...