IP ממומן על ידי מדעי הטבע והנדסה מועצת המחקר של מענק קנדה (NSERC # 418233-2012); פונדס דה Recherche du Québec-Santé (FRQS), פרס קריירה לסרטן השד של קוובק, ומענק למנהיגים מייסד הקרן הקנדית לחדשנות. א.ד. קיבל מלגה מארגון ארנדנד-פריייר.

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למחברים אין מה להכריז.

תאים תאים אינטראקציות באמצעות צמתים נדרשים לתפקוד תקין ופיתוח של איברים רבים, כגון בלוטת החלב. מחקרים הראו כי חלבונים פונקציונליים יכולים לווסת את הפונקציה ואת היציבות של אחד אחר ולהפעיל transduction האות על ידי קשירה זה לזה בקרום התא 10 . הפרוטוקולים שהוצגו ב?...

התפתחות בלוטת החלב ופיתוח מתרחשת בעיקר לאחר הלידה. איבר זה מתחדד ללא הרף לאורך כל חיי הפוריות של יונק 1 . האפיתל בבלוטת החלב הבוגרת מורכבת משכבה פנימית של תאי אפיתל לומינליים ושכבה חיצונית של תאים ביזליים, המורכבים בעיקר מתאי מיופתיטליים, מוקפים בקרום מרתף 2 . לבדיקה טובה על מבנה בלוטת החלב ופיתוח, הקורא יכול להתייחס Sternlicht 1 . תאים תא אינטראקציות דרך הפער (GJ), הדוק (TJ), והצמדות (AJ) צמתים הדרושים להתפתחות תקינה ותפקוד של בלוטה 1 , 3 , 4 , 5 , 6 . המרכיבים העיקריים של צמתים אלה בלוטת החלב Murine הם Cx26, Cx30, Cx32, ו Cx43 (GJ); קלודין -1, -3, -4, -7 וZO-1 (TJ); ו E-cadherin, P-cadherin, ו β-catenin (AJ) 7 , 8 . רמות הביטוי של אלה חלבונים ג &#39;וניקט שונים להשתנות באופן תלוי שלב במהלך התפתחות בלוטת החלב, מה שמציע אינטראקציה תא תא אינטראקציה הדרישות 9 . GJ, TJ ו- AJ קשורים באופן מבניים ופונקציונליים ומקיפים חלבונים מבניים או רגולטוריים אחרים לאתרים הסמוכים של תאים סמוכים, ובכך יוצרים קשר גנטי. ההרכב של הקשר הג&#39;ונקטי יכול להשפיע על גישור עם cytoskeleton הבסיסית, כמו גם חדירות ויציבות nexus, ולכן יכול להשפיע על הפונקציה של בלוטת 8 , 9 , 10 , 11 . המרכיבים של צמתים בין תאים המתגוררים nexuses פונקציונלי או אינטראקציה אחד עם השני ב difבשלבים התפתחותיים של התפתחות בלוטת החלב נותחו לאחרונה באמצעות שיתוף immunofluorescence (שיתוף IF) ושיתוף אימונופרסיפיטיון (שיתוף IP) 9 . בעוד טכניקות אחרות מאפשרות להעריך את הקשר הפונקציונלי בין חלבונים, שיטות אלו אינן מוצגות בכתב היד הזה. כאשר חלבונים פועלים רק לבדם לתפקוד, לימוד אינטראקציות בין חלבונים לחלבון (PPI), כגון טרנספורמציות אותות ומפליות ביוכימיות, הוא חיוני לחוקרים רבים ויכול לספק מידע משמעותי על תפקוד החלבונים. Co-IF וניתוח מיקרוסקופי לעזור להעריך כמה חלבונים החולקים את אותו חלל תת-תאי. עם זאת, מספר מטרות מוגבל על ידי נוגדנים, אשר חייב להיות הרים בעלי חיים שונים, ועל ידי גישה מיקרוסקופ confocal מצויד לייזרים שונים אורך גלאי ספקטרלי עבור ריבוב. Co-IP מאשרת או מגלה זיקה גבוהה פיזית אינטראקציות betwEen שני חלבונים או יותר המתגוררים בתוך קומפלקס חלבון. למרות הפיתוח של טכניקות חדשניות, כגון העברת אנרגיה תהודה הקרינה (סריג) 12 ו assay קשירת הקרבה (PLA) 13 , אשר יכול לזהות בו זמנית את לוקליזציה ואינטראקציות של חלבונים, שיתוף IP נשאר טכניקה נאותה ובמחיר סביר ללמוד אינטראקציות בין חלבונים אנדוגניים. שיטת צעד אחר צעד המתוארת בכתב יד זה יקל על המחקר של לוקליזציה חלבון PPIs להצביע על מלכודות להימנע בעת חקר PPIs אנדוגני בבלוטות החלב. המתודולוגיה מתחילה בהצגת הליכי השימור השונים לרקמות הנדרשות לכל טכניקה. חלק 1 מציג כיצד ללמוד חלבון שיתוף לוקליזציה בשלושה שלבים: 1) חתך של בלוטות החלב, ii) פעמיים או משולשת תיוג של חלבונים שונים באמצעות טכניקה שיתוף IF, ו- iii) הדמיה שללוקליזציה של חלבונים. חלק 2 מראה כיצד להאיץ חלבון אנדוגני ולזהות חלבונים אינטראקציה שלה בשלושה שלבים: i) הכנה lysate, ii) immunoprecipitation חלבון עקיף, ו- iii) זיהוי שותף מחייב על ידי SDS-PAGE כתם המערבי. כל צעד של פרוטוקול זה הוא מותאם עבור מכרסמים בלוטות הבלוטות החלב ומייצר באיכות גבוהה, ספציפי, תוצאות לשחזור. פרוטוקול זה יכול לשמש גם כנקודת מוצא ללימודי PPI ברקמות אחרות או שורות תאים.

<table><tbody><tr><td>Mice strain and stage</td><td>St. Constant, Quebec, Canada</td><td></td><td>C57BL/6 Femals; pregnancy day 18 (P18) and lactation day 14 (L14), Charles River Canada&amp;nbsp;</td></tr><tr><td>PBS 10x (stock)</td><td></td><td></td><td>1) Dissolve 80 g NaCl (F.W.: 58.44), 2 g KCl (F.W. 74.55), 26.8 g Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;#183;7H&lt;sub&gt;2&lt;/sub&gt;O (F.W. 268.07) and 2.4 g KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; (F.W.:136.09) in 800 mL distilled water;&lt;br/&gt; 2) Adjust the PH to 7.4;&lt;br/&gt; 3) Add water to reach to the 1 L final volume.</td></tr><tr><td>TBS 10x (stock)</td><td></td><td></td><td>1) Dissolve 60.5 g TRIS, 87.6 g NaCl in 800 mL distilled water;&lt;br/&gt;2) Adjust the pH to 7.5;&lt;br /&gt;3) Add water to reach to the 1 L final volume.</td></tr><tr><td colspan="4">&lt;strong&gt;Part 1: Immunofluorescence&lt;/strong&gt;</td></tr><tr><td>Freezing media</td><td>VWR International, Ville Mont-Royal, QC, Canada</td><td>95067-840</td><td>VMR frozen sections compound&amp;nbsp;</td></tr><tr><td>Microtome</td><td>Mississauga, ON, Canada</td><td>956640</td><td>Microm HM525, Thermo fisher scientific HM525 NX Cryostat 115 V 60 Hz</td></tr><tr><td>Blades</td><td>C.L. Sturkey, Inc. Les Produits&amp;nbsp;Scientifiques&amp;nbsp;ESBE&amp;nbsp;St-Laurent, QC, Canada</td><td>BLM1001C</td><td>High profile gold coated blades</td></tr><tr><td>Pap pen</td><td>Cedarlane, Burlington, ON, Canada</td><td>8899</td><td>Super PAP Pen, Thermo fisher scientific</td></tr><tr><td>Microscopic slides</td><td>Fisher Scientific, Burlington, ON, Canada</td><td>12-550-15</td><td>Fisherbrand&amp;nbsp;Superfrost Plus Microscope Slides</td></tr><tr><td>Formaldehyde</td><td>BioShop Canada Inc, Burlington, ON, Canada</td><td>FOR201.1</td><td>Forlmadehyde</td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Santa Cruz Biotechnology, Inc, California, USA</td><td></td><td></td></tr><tr><td>Blocking solution</td><td></td><td></td><td>3% BSA in TBS</td></tr><tr><td>Wash solution</td><td></td><td></td><td>TBS-Tween 20 0.1%</td></tr><tr><td>Polysorbate 20</td><td>Oakville, ON, Canada</td><td>P 9416</td><td>Tween 20, Sigma-Aldrich</td></tr><tr><td>Mounting media</td><td>Cedarlane, Burlington, ON</td><td>17984-25(EM)</td><td>Fluoromount-G</td></tr><tr><td>First &amp;amp; secondary antibodies</td><td>Cell Signaling, Beverly, MA, USA</td><td>See Comments</td><td>E-Cadherin (4A2) Mouse mAb (#14472s) 1/50 (Cell Signaling) with anti-mouse IgG Fab2 Alexa Fluor 555 (#4409s), Cell Signaling&amp;nbsp;</td></tr><tr><td>First &amp;amp; secondary antibodies&amp;nbsp;</td><td>Life technologies, Waltham, MA, USA &amp;amp; Cell Signaling, Beverly, MA, USA</td><td>See Comments</td><td>Claudin-7 (#34-9100) 1/100 (Life Technologies) with anti-rabbit IgG Fab2 Alexa Fluor 488 (#4412s) (Cell Signaling)&amp;nbsp;</td></tr><tr><td>First &amp;amp; secondary antibodies&amp;nbsp;</td><td>Santa Cruz Biotechnology, Inc, California, USA; Fischer Scientific, Burlington, ON, Canada&amp;nbsp;</td><td>See Comments</td><td>&amp;beta;-Catenin Antibody (C-18): sc-1496 (SANTA CRUZ) with anti-Goat IgG (H+L) Alexa Fluor 568 (#A11057), Molecular Probe (Fisher Scientific)</td></tr><tr><td>First &amp;amp; secondary antibodies&amp;nbsp;</td><td>Life technologies, Waltham, MA, USA &amp;amp; Cell Signaling, Beverly, MA, USA</td><td>See Comments</td><td>Connexin26&amp;nbsp; (#33-5800) 1/75 (Life Technologies) with anti-mouse IgG Fab2 Alexa Fluor 647 (#4410s)&amp;nbsp;</td></tr><tr><td>Nuclei stain</td><td>Fisher Scientific, Burlington, ON, Canada</td><td>D1306</td><td>DAPI (4&#039;,6-Diamidino-2-Phenylindole, Dihydrochloride) 1/1,000 in PBS</td></tr><tr><td>Fluorescent microscope</td><td>Nikon Canada, Mississauga, On, Canada</td><td></td><td>Nikon A1R+ confocal microscopic laser equipped with a spectral detector&amp;nbsp;</td></tr><tr><td>Software of IF images analysis</td><td>Nikon Canada, Mississauga, On, Canada</td><td></td><td>NIS-elements software (version 4)</td></tr><tr><td colspan="4">&lt;strong&gt;Part 2: Immunoprecipitation&lt;/strong&gt;</td></tr><tr><td>Triple-detergent Lysis buffer (100&amp;nbsp;mL) pH=8.0</td><td></td><td></td><td>1) Mix 50 mM TRIS (F.W.: 121.14), 150&amp;nbsp;mM NaCl (F.W.: 58.44), 0.02% Sodium Azide, 0.1% SDS, 1% NONIdET P40, 0.5% Sodium Deoxycholate in 80 mL distilled H&lt;sub&gt;2&lt;/sub&gt;O.&lt;br/&gt; 2) Adjust the pH to 8.0 with HCl 6&amp;nbsp;N (~0.5&amp;nbsp;mL).&lt;br/&gt; 3) Adjust the volume to 100&amp;nbsp;mL. Keep it in fridge.&lt;br/&gt; At the day of protein extraction, use 1/100 NaVo&lt;sub&gt;3&lt;/sub&gt;, 1/100 protease/phosphatase inhibitor and 1/25 NAF in calculated amount of Triple detergent lysis buffer:&lt;br/&gt; Sodium Fluoride (stock) solution 1.25&amp;nbsp;M (F.W.: 41.98), Sodium Orthovanadate (stock) Solution 1&amp;nbsp;M (F.W.: 183.9)</td></tr><tr><td>Protease/phosphatase inhibitor</td><td>Fisher Scientific, Burlington, ON</td><td>78441</td><td>Halt Protease and Phosphatase Inhibitor Cocktail, EDTA-free (100x)</td></tr><tr><td>Protein dosage</td><td>Thermo Scientific, Rockford, Illinois, USA</td><td>23225</td><td>Pierce BCA protein assay kit&amp;nbsp;</td></tr><tr><td>Tissue grinder</td><td>Fisher Scientific, Burlington, ON</td><td>FTH-115</td><td>Power 125, Model FTH-115</td></tr><tr><td>Magnetic beads and stand</td><td>Millipore, Etobicoke, ON, Canada</td><td></td><td>PureProteome Protein G Magnetic Bead System (LSKMAGG02)</td></tr><tr><td>Wash solution for IP</td><td></td><td></td><td>PBS or PBS-Tween20 0.1% depending to the step</td></tr><tr><td>Primary antibodies for immunoprecipitation</td><td>Cell Signaling, Beverly, MA, USA</td><td>See Comments</td><td>IgG Rabbit (rabbit (DA1E) mAb IgG Isotype control (#3900s) (Cell Signaling) 0.5 &amp;micro;L/200 &amp;micro;L</td></tr><tr><td>Primary antibodies for immunoprecipitation</td><td>Cell Signaling, Beverly, MA, USA</td><td>See Comments</td><td>IgG Mouse mouse (G3A1) mAb IgG Isotype control (#5415s) (Cell Signaling) 0.5 &amp;micro;L/200 &amp;micro;L</td></tr><tr><td>Primary antibodies for immunoprecipitation</td><td>Sigma-Aldrich, Oakville, ON, Canada</td><td>See Comments</td><td>Connexin43 (#C6219) (Sigma-Aldrich) 4&amp;nbsp;&amp;micro;L/200&amp;nbsp;&amp;micro;L</td></tr><tr><td>Primary antibodies for immunoprecipitation</td><td>Cell Signaling, Beverly, MA, USA</td><td>See Comments</td><td>E-cadherin (4A2) Mouse mAb (#14472s) (Cell Signaling) 1 &amp;micro;L/200 &amp;micro;L</td></tr><tr><td>Laemmli buffer&amp;nbsp;</td><td>BIO-RAD, Mississauga, Ontario, Canada</td><td>1610747</td><td>4x Laemmli Sample Buffer (Add &amp;beta;-mercaptoethanol following manufacturer recommendation)</td></tr><tr><td>Acidic glycine&amp;nbsp;</td><td>Fisher Scientific, Burlington, ON</td><td>PB381-5</td><td>0.2 M glycine; adjust pH=2.5 with HCl&amp;nbsp;</td></tr><tr><td>Tris&amp;nbsp;</td><td>Fisher Scientific, Burlington, ON</td><td>BP152-1</td><td>1 M (pH=8)&amp;nbsp;</td></tr><tr><td>SDS-PAGE acrylamide gels&amp;nbsp;</td><td>BIO-RAD, Mississauga, ON, Canada</td><td>1610180 -5</td><td>TGX Stain-Free FastCast Acrylamide Solutionss (7.8%, 10%, 12%)</td></tr><tr><td>Running buffer 10x&amp;nbsp;</td><td>BIO-RAD, Mississauga, ON, Canada</td><td>1704272</td><td>Tris 30.3&amp;nbsp;g/glycine 144.1&amp;nbsp;g /SDS 10&amp;nbsp;g in 1 L distilled water</td></tr><tr><td>Membranes</td><td>BIO-RAD, Mississauga, ON, Canada</td><td>1704272</td><td>PVDF membranes, Trans-Blot Turbo RTA Mini PVDF Transfer Kit</td></tr><tr><td>Transfer method</td><td>BIO-RAD, Mississauga, ON, Canada</td><td>1704155</td><td>Trans-Blot Turbo Transfer System</td></tr><tr><td>Dry Milk</td><td>Smucker Food of Canada Co, Markham, ON, Canada</td><td></td><td>Fat Free Instant Skim Milk Powder, Carnation</td></tr><tr><td>Blocking solution for blots</td><td></td><td></td><td>5% dry milk in TBS-Tween 20 0.1%</td></tr><tr><td>Washing solutions for blots</td><td></td><td></td><td>TBS-Tween 20 0.1%</td></tr><tr><td>Primary and secondary antibodies for blots (10&amp;nbsp;mL)</td><td>Sigma-Aldrich, Oakville, Ontario &amp;amp; Abcam, Toronto, ON, Canada</td><td>See Comments</td><td>Connexin43 (#C6219) (Sigma-Aldrich) 1/2,500 with HRP-conjugated Veriblot for IP secondary antibody (ab131366) 1/5,000 (Abcam, Toronto, ON, Canada)</td></tr><tr><td>Primary and secondary antibodies for blots (10&amp;nbsp;mL)</td><td>Cell Signaling, Beverly, MA, USA &amp;amp; Abcam, Toronto, ON, Canada</td><td>See Comments</td><td>E-cadherin (24E10) rabbit mAb 1/1,000 (#3195s) (Cell Signaling) 1/1,000 with HRP-conjugated Veriblot for IP secondary antibody (ab131366) 1/5,000 (Abcam, Toronto, ON, Canada)</td></tr><tr><td>Primary and secondary antibodies for blots (10&amp;nbsp;mL)</td><td>Life technologies, Waltham, MA, USA &amp;amp; Abcam, Toronto, ON, Canada</td><td>See Comments</td><td>Claudin-7 (#34-9100) (Life technologies) 1/1,000 with HRP-conjugated Veriblot for IP secondary antibody (ab131366) 1/5,000 (Abcam, Toronto, ON, Canada)</td></tr><tr><td>Primary and secondary antibodies for blots (10&amp;nbsp;mL)</td><td>Life technologies, Waltham, MA, USA &amp;amp; Abcam, Toronto, ON, Canada</td><td>See Comments</td><td>Claudin3 (#34-1700) (Life technologies) 1/1,000 with HRP-conjugated Veriblot for IP secondary antibody (ab131366) 1/5,000 (Abcam, Toronto, ON, Canada)</td></tr><tr><td>Luminol solution for signal detection on blots</td><td>BIO-RAD, Mississauga, ON, Canada</td><td>1705061</td><td>Clarity Western ECL Blotting Substrate</td></tr><tr><td>Imaging blots</td><td>BIO-RAD, Mississauga, ON, Canada</td><td>1708280</td><td>ChemiDoc MP imaging system</td></tr><tr><td>Analayzing blots</td><td>BIO-RAD, Mississauga, ON, Canada</td><td></td><td>ImageLab 5.2 software&amp;nbsp;</td></tr></tbody></table>

analysis of protein-protein interactions and co-localization between components of gap, tight, and adherens junctions in murine mammary glands

אינטראקציות תא סלולרי תפקיד מרכזי בשמירה על שלמות הרקמה ואת המכשול בין תאים שונים של בלוטת החלב. אינטראקציות אלה מסופקות על ידי חלבונים גנטיים המרכיבים נקסוס בין תאים סמוכים. מיצוקלציה של חלבון פונקציונאלי וירידה באסוציאציות פיזיות עם השותפים המחייבים שלהם עלולה לגרום לאובדן תפקוד, וכתוצאה מכך לתפקוד איברים. לכן, זיהוי לוקליזציה של חלבונים וחלבוני חלבונים (PPI) ברקמות הרגילות והמחלות, חיוניים למציאת עדויות ומנגנונים חדשים המביאים להתפתחות מחלות או שינויים במעמד ההתפתחותי. כתב היד מציג שיטה דו-שלבית להערכת PPIs בבלוטות החלב של Murine. בפרוטוקול סעיף 1, שיטה לבצע co- אימונופלורסנציה (שיתוף IF) באמצעות נוגדנים שהועלו נגד חלבונים של עניין, ואחריו נוגדנים משניים שכותרתו עם fluorochromes, מתואר. למרות שיתוף שיתוף IFבגלל ההוכחה של קרבת החלבונים, היא מאפשרת ללמוד את האינטראקציות הגופניות שלהם. לכן, פרוטוקול מפורט עבור שיתוף immunoprecipitation (שיתוף IP) מסופק בסעיף פרוטוקול 2. שיטה זו משמשת כדי לקבוע את האינטראקציות הגופניות בין חלבונים, מבלי לאשר אם אינטראקציות אלה הן ישירות או עקיפות. בשנים האחרונות, Co-IF ו- co-IP טכניקות הוכיחו כי רכיבים מסוימים של צמתים בין תאגידים שיתוף וליצור אינטראקציה יחד, יצירת תלויי שלב nexuses הגדילה המשתנים במהלך התפתחות בלוטת החלב.

כדי לקבוע אם GJ, AJ, ו TJ רכיבים יכולים לתקשר יחד בלוטת החלב, co-IF מבחני בוצעו לראשונה. Cx26, חלבון GJ, ו- β-Catenin, חלבון AJ, נבדקו באמצעות נוגדנים ספציפיים וגילו באמצעות נוגדנים של עכבר פלואורופורי-מצומדות (647, ירוק, pseudocolor) ועז-568 (אדום), בהתאמה ( איור 1B ו- <st...

Watch this Scientific Journal Video about Analysis of Protein-protein Interactions and Co-localization Between Components of Gap, Tight, and Adherens Junctions in Murine Mammary Glands at JoVE.com

Analysis of Protein-protein Interactions and Co-localization Between Components of Gap, Tight, and Adherens Junctions in Murine Mammary Glands

צמתים בין-תאיים הם דרישות עבור פונקציות ספציפיות של בלוטת החלב והתפתחותן. כתב היד מספק פרוטוקול מפורט לחקר האינטראקציות בין חלבונים לחלבון (PPI) לבין שיתוף לוקליזציה באמצעות בלוטות החלב Murine. טכניקות אלה מאפשרות לחקור את הדינמיקה של הקשר הפיזי בין צמתים בין-תאיים בשלבי התפתחות שונים.

ניתוח אינטראקציות חלבון חלבון שיתוף לוקליזציה בין רכיבי פערים, צמודים, ו adherens צמתים Murine בלוטות החלב

Cell-cell interactions play a pivotal role in preserving tissue integrity and the barrier between the different compartments of the mammary gland. These ...

analysis-protein-protein-interactions-co-localization-between

Research

JoVE Journal

Developmental Biology

10.0K Views.  INRS, Institut Armand-Frappier.  צמתים בין-תאיים הם דרישות עבור פונקציות ספציפיות של בלוטת החלב והתפתחותן. כתב היד מספק פרוטוקול מפורט לחקר האינטראקציות בין חלבונים לחלבון (PPI) לבין שיתוף לוקליזציה באמצעות בלוטות החלב Murine. טכניקות אלה מאפשרות לחקור את הדינמיקה של הקשר הפי...

Video: Analysis of Protein-protein Interactions and Co-localization Between Components of Gap, Tight, and Adherens Junctions in Murine Mammary Glands

Sectioning Mammary Gland Whole Mounts for Lesion Identification

Normal mammary gland development may be altered by exposure to environmental toxicants and pharmaceutical products, excessive exposure to hormones, and genetic alterations. Mammary gland whole mounts are an inexpensive method to capture the progression of morphological changes that may arise after exposure. However, in later life, when abnormalities are more prone to develop, sole reliance on this one method may not always provide enough information to make a proper diagnosis of the abnormality. Historically, in chemical test guideline studies, a single mammary gland is removed at necropsy and prepared as a hematoxylin and eosin (H&#38;E)-stained section. The incorporation of contralateral mammary whole-mount collection and analysis decreases the likelihood of a false-negative assessment. Evaluation of the whole mount is limited by the presence of one or two entire mammary glands on a slide, and in some cases, the abnormalities observed in the whole mount are not uniformly represented in the H&#38;E section.&#160; The goal of this study was to develop a protocol for converting coverslipped mammary whole mounts to H&#38;E-stained sections so that lesions that would otherwise have been missed or that are difficult to diagnose can be identified. Here, we detail a method to produce a high-quality, paraffin-embedded H&#38;E section from a mammary gland that was initially prepared as a whole mount. In comparison to a tissue that was intentionally prepared for H&#38;E sectioning, the whole mount requires additional preparation for tissue removal and processing. However, this method is considered inexpensive, as it requires common lab reagents and little additional time. As a result, this method can provide invaluable information on how chemical and environmental exposures alter normal mammary development, as well as display changes that occur because of genetic modifications.

We developed a method to successfully remove, process, section, and stain, for histopathological evaluation, mammary tissue that had originally been fixed on slides as whole mounts. This method may promote the collection and evaluation of mammary gland whole mounts in reproductive and developmental test guideline studies.

חתך בלוטות החלב שלמות מלא עבור זיהוי הנגע

Normal mammary gland development may be altered by exposure to environmental toxicants and pharmaceutical products, excessive exposure to hormones, and ...

Cancer Research

Differentiation of Mouse Breast Epithelial HC11 and EpH4 Cells

Cadherins play an important role in the regulation of cell differentiation as well as neoplasia. Here we describe the origins and methods of the induction of differentiation of two mouse breast epithelial cell lines, HC11 and EpH4, and their use to study complementary stages of mammary gland development and neoplastic transformation.
The HC11 mouse breast epithelial cell line originated from the mammary gland of a pregnant Balb/c mouse. It differentiates when grown to confluence attached to a plastic Petri dish surface in medium containing fetal calf serum and Hydrocortisone, Insulin and Prolactin (HIP medium). Under these conditions, HC11 cells produce the milk proteins &#946;-casein and whey acidic protein (WAP), similar to lactating mammary epithelial cells, and form rudimentary mammary gland-like structures termed &#34;domes&#34;.
The EpH4 cell line was derived from spontaneously immortalized mouse mammary gland epithelial cells isolated from a pregnant Balb/c mouse. Unlike HC11, EpH4 cells can fully differentiate into spheroids (also called mammospheres) when cultured under three-dimensional (3D) growth conditions in HIP medium. Cells are trypsinized, suspended in a 20% matrix consisting of a mixture of extracellular matrix proteins produced by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, plated on top of a layer of concentrated matrix coating a plastic Petri dish or multiwell plate, and covered with a layer of 10% matrix-containing HIP medium. Under these conditions, EpH4 cells form hollow spheroids that exhibit apical-basal polarity, a hollow lumen, and produce &#946;-casein and WAP.
Using these techniques, our results demonstrated that the intensity of the cadherin/Rac signal is critical for the differentiation of HC11 cells. While Rac1 is necessary for differentiation and low levels of activated RacV12 increase differentiation, high RacV12 levels block differentiation while inducing neoplasia. In contrast, EpH4 cells represent an earlier stage in mammary epithelial differentiation, which is inhibited by even low levels of RacV12.

We describe techniques for differentiation induction of two breast epithelial lines, HC11 and EpH4. While both require fetal calf serum, insulin, and prolactin to produce milk proteins, EpH4 cells can fully differentiate into mammospheres in three-dimensional culture. These complementary models are useful for signal transduction studies of differentiation and neoplasia.

הבידול של השד העכבר האפיתל HC11 ו EpH4 תאים

Cadherins play an important role in the regulation of cell differentiation as well as neoplasia. Here we describe the origins and methods of the induction ...

Quantifying Branching Density in Rat Mammary Gland Whole-mounts Using the Sholl Analysis Method

An increasing number of studies are utilizing the rodent mammary gland as an endpoint for assessing the developmental toxicity of a chemical exposure. The effects these exposures have on mammary gland development are typically evaluated using either basic dimensional measurements or by scoring morphological characteristics. However, the broad range of methods for interpreting developmental changes could lead to inconsistent translations across laboratories. A common method of assessment is needed so that proper interpretations can be formed from data being compared across studies. The present study describes the application of the Sholl analysis method to quantify mammary gland branching characteristics. The Sholl method was originally developed for use in quantifying neuronal dendritic patterns. By using ImageJ, an open-source image analysis software package, and a plugin developed for this analysis, the mammary gland branching density and the complexity of a mammary gland from a peripubertal female rat were determined. The methods described here will enable the use of the Sholl analysis as an effective tool for quantifying an important characteristic of mammary gland development.

Mammary gland development in the rodent has typically been evaluated using descriptive assessments or by measuring basic physical attributes. Branching density is an indicator of mammary development that is difficult to quantify objectively. This protocol describes a reliable method for the quantitative assessment of mammary gland branching characteristics.

כימות צפיפות הענף בבלוטת החלב עכברוש- mounts כולו באמצעות שיטת ניתוח Sholl

An increasing number of studies are utilizing the rodent mammary gland as an endpoint for assessing the developmental toxicity of a chemical exposure. The ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

הנדסה תלת מימדי רקמות אפיתל Embedded בתוך מטריקס

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Bioengineering

Generation of Mosaic Mammary Organoids by Differential Trypsinization

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine mammary gland is composed of two distinct epithelial cell compartments, serving different functions: the outer, contractile myoepithelial compartment and the inner, secretory luminal compartment. Here, we describe a method by which the cells comprising these compartments are isolated and then combined to investigate their individual lineage contributions to mammary gland morphogenesis and differentiation. The method is simple and efficient and does not require sophisticated separation technologies such as fluorescence activated cell sorting. Instead, we harvest and enzymatically digest the tissue, seed the epithelium on adherent tissue culture dishes, and then use differential trypsinization to separate myoepithelial from luminal cells with ~90% purity. The cells are then plated in an extracellular matrix where they organize into bilayered, three-dimensional (3D) organoids that can be differentiated to produce milk after 10 days in culture. To test the effects of genetic mutations, cells can be harvested from wild type or genetically engineered mouse models, or they can be genetically manipulated prior to 3D culture. This technique can be used to generate mosaic organoids that allow investigation of gene function specifically in the luminal or myoepithelial compartment.

The mammary gland is a bilayered structure, comprising outer myoepithelial and inner luminal epithelial cells. Presented is a protocol to prepare organoids using differential trypsinization. This efficient method allows researchers to separately manipulate these two cell types to explore questions concerning their roles in mammary gland form and function.

הדור של הפסיפס של חלב באמצעות טריסיזציה דיפרנציאלית

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine ...

Evaluation of Mammary Gland Development and Function in Mouse Models

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

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

הערכת התפתחות בלוטת החלב פונקציה ועל מודלים עכבר

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

Biology

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

Histology volume reconstruction facilitates the study of 3D shape and volume change of an organ at the level of macrostructures made up of cells. It can also be used to investigate and validate novel techniques and algorithms in volumetric medical imaging and therapies. Creating 3D high-resolution atlases of different organs1,2,3 is another application of histology volume reconstruction. This provides a resource for investigating tissue structures and the spatial relationship between various cellular features. We present an image registration approach for histology volume reconstruction, which uses a set of optical blockface images. The reconstructed histology volume represents a reliable shape of the processed specimen with no propagated post-processing registration error. The Hematoxylin and Eosin (H&#38;E) stained sections of two mouse mammary glands were registered to their corresponding blockface images using boundary points extracted from the edges of the specimen in histology and blockface images. The accuracy of the registration was visually evaluated. The alignment of the macrostructures of the mammary glands was also visually assessed at high resolution.
This study delineates the different steps of this image registration pipeline, ranging from excision of the mammary gland through to 3D histology volume reconstruction. While 2D histology images reveal the structural differences between pairs of sections, 3D histology volume provides the ability to visualize the differences in shape and volume of the mammary glands.

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

שחזור נפח היסטולוגיה 3 ממדים ויישומו לחקר מאוס החלב בלוטות

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

Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple method for the immune detection of proteins, the mouse lactating mammary gland being taken as an example. A protocol for the preparation of the tissue samples, especially concerning the dissection of mouse mammary gland, tissue fixation and frozen tissue sectioning, are detailed. A standard protocol to perform indirect immunofluorescence, including an optional antigen retrieval step, is also presented. The observation of the labeled tissue sections as well as image acquisition and post-treatments are also stated. This procedure gives a full overview, from the collection of animal tissue to the cellular localization of a protein. Although this general method can be applied to other tissue samples, it should be adapted to each tissue/primary antibody couple studied.

The indirect immunofluorescence protocol described in this article allows the detection and the localization of proteins in the mouse mammary gland. A complete method is given to prepare mammary gland samples, to perform immunohistochemistry, to image the tissue sections by fluorescence microscopy, and to reconstruct images.

Immunofluorescence עקיף על סעיפים קפואים של עכבר בלוטת החלב

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple ...

Isolation of Intact, Whole Mouse Mammary Glands for Analysis of Extracellular Matrix Expression and Gland Morphology

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal architecture. Here, a small pocket below the skin was created using Mayo scissors, allowing separation of the glands within the subcutaneous tissue from the underlying peritoneum. Visualization of the glands was aided by the use of 3.5x-R surgical micro loupes. The pelt was inverted and pinned back allowing identification of the intact mammary fat pads. Each of the #4 abdominal glands was bluntly dissected by sliding the scalpel blade laterally between the subcutaneous layer and the glands. Immediately post-harvest, glands were placed in 10% neutral buffered formalin for subsequent tissue processing. Excision of the entire gland is advantageous because it primarily eliminates the risk of excluding important tissue-wide interactions between ductal epithelial cells and other microenvironmental cellular populations that could be missed in a partial biopsy. One drawback of the methodology is the use of serial sections from fixed tissues which limits analyses of ductal morphogenesis and protein expression to discrete locations within the gland. As such, changes in ductal architecture and protein expression in 3 dimensions (3D) is not readily obtainable. Overall, the technique is applicable to studies requiring whole intact murine mammary glands for downstream investigations such as developmental ductal morphogenesis or breast cancer.

Here, we present a protocol for the isolation of whole, intact mouse mammary glands to investigate extracellular matrix (ECM) expression and ductal morphology. Mouse #4 abdominal glands were extracted from 8-10 week old female nulliparous mice, fixed in neutral buffered formalin, sectioned and stained using immunohistochemistry for ECM proteins.

בידוד של בלוטות מוגדלות ללא פגע, כל עכבר לניתוח של בלוטת מורפולוגיה וביטוי מטריצה חוץ-תאית

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal ...

Longitudinal Intravital Microscopy Using a Mammary Imaging Window with Replaceable Lid

The branched structure of the mammary gland is highly dynamic and undergoes several phases of growth and remodeling after birth. Intravital microscopy in combination with skin flap surgery or implantation of imaging windows has been used to study the dynamics of the healthy mammary gland at different developmental stages. Most mammary imaging technologies are limited to a time frame of hours to days, whereas the majority of mammary gland remodeling processes occur in time frames of days to weeks. To study mammary gland remodeling, methods that allow optical access to the tissue of interest for extended time frames are required. Here, an improved version of the titanium mammary imaging window with a replaceable lid (R.MIW) is described that allows high-resolution imaging of the mammary gland with a cellular resolution for up to several weeks. Importantly, the R.MIW provides tissue access over the entire duration of the intravital imaging experiment and could therefore be used for local tissue manipulation, labeling, drug administration, or image-guided microdissection. Taken together, the R.MIW enables high-resolution characterization of the cellular dynamics during mammary gland development, homeostasis, and disease.

This protocol describes a novel mammary imaging window with a replaceable lid (R.MIW). Intravital microscopy after implantation of the R.MIW allows for longitudinal and multi-day imaging of the healthy and diseased mammary gland with a cellular resolution during the different developmental stages.

מיקרוסקופיה תוך-ורידית אורכית באמצעות חלון הדמיה של יונקים עם מכסה הניתן להחלפה

The branched structure of the mammary gland is highly dynamic and undergoes several phases of growth and remodeling after birth. Intravital microscopy in ...