Phospho-אפיטופים Immunostaining באיברים ריסי של עוברי דג הזברה שלמים הר
Summary
טכניקות מתוארים כדי immunostain-אפיטופים phospho כולן עוברי דג הזברה ולאחר מכן לנהל לוקליזציה confocal ניאון בשני צבעים במבנים הסלולר קטן כמו cilia העיקרי. טכניקות לתיקון והדמיה יכול להגדיר את המיקום ואת קינטיקה של המראה או הפעלה של חלבונים ספציפיים.
Abstract
ההתפשטות מהירה של תאים, ביטוי ספציפי הרקמות של גנים ואת הופעתה של רשתות איתות לאפיין התפתחות עוברית מוקדמת של כל בעלי החוליות. קינטיקה ומיקום של אותות – אפילו בתוך תאים בודדים – בעובר המתפתח משלים זיהוי של גנים התפתחותיים חשובים. טכניקות Immunostaining מתוארים כי הוכחו להגדיר את קינטיקה של אותות בעלי חיים תאיים ושלמים במבנים קטנים כמו cilia העיקרי. טכניקות לתיקון, הדמיה ועיבוד תמונות באמצעות מיקרוסקופ מתחם confocal-סריקת לייזר ניתן להשלים כמה כמו 36 שעות.
דג הזברה (Danio rerio) הוא אורגניזם רצוי חוקרים המבקשים לבצע מחקרים בתוך בעלי החוליות היא הזולה רלוונטי מחלות אנושיות. knockouts או knockdowns הגנטיים חייב להיות מאושר על ידי האובדן של מוצר החלבון בפועל. אישור כזה של אובדן חלבוןיכול להיות מושג באמצעות הטכניקות המתוארות כאן. רמזים לתוך מסלולי איתות ניתן לפענח גם באמצעות נוגדנים כי הם תגובתי עם חלבונים ששונו שלאחר translationally ידי זרחון. שמירה ואופטימיזציה של המדינה פוספורילציה של אפיטופ לכן זה קריטי כדי קביעה זו לבין מושגת על ידי פרוטוקול זה.
מחקר זה מתאר שיטות לתקן עוברים במהלך 72 שעות של פיתוח הראשון שיתוף בתרגום במגוון אפיטופים רלוונטי עם cilia בתוך שלפוחיות (KV) של Kupffer, הכליה ואת האוזן הפנימית. טכניקות אלו הם ישירים, אינם דורשים דיסקציה יכול להסתיים בתוך פרק זמן קצר יחסית של זמן. הקרנת ערימות תמונת confocal לתוך תמונה אחת היא אמצעי שימושי של הצגת נתונים אלה.
Introduction
The techniques described here are the outcome of studies that have sought to define downstream targets of Ca2+ signals during events that occur during early development, including fertilization, gastrulation, somitogenesis and trunk, eye, brain and organ formation.1-3 The original discoveries of embryonic Ca2+ signaling were dependent on the use of natural and engineered Ca2+ indicators, such as aequorin4 and fura-2.5 Even with current technology, the detection of transient elevations of Ca2+ requires cumbersome analytical tools and does not reveal the targets of such Ca2+ signals.
This laboratory investigates Ca2+ signals that act through the Ca2+/calmodulin-dependent (multifunctional) protein kinase known as CaMK-II, an enzyme that is enriched in the central nervous system and originally identified as a regulator of long-term potentiation.6 CaMK-II is not brain-specific, is widely expressed and highly conserved throughout the entire lifespan and bodies of species throughout the animal kingdom, including invertebrates.7,8 CaMK-II has the unique capability of sustaining its own activity even after Ca2+ levels have diminished due to its ability to autophosphorylate at Thr287. In this autophosphorylated state, CaMK-II remains active in a Ca2+/CaM-independent manner, until dephosphorylated.6 Thus, the localization of phosphorylated CaMK-II (Thr287) can identify cells in which natural, relevant Ca2+ elevations have occurred.
An antibody against autophosphorylated (P-Thr287) mammalian CaMK-II has been well-characterized and was initially used to localize activated CaMK-II in brain tissue.9 Zebrafish (Danio rerio) have seven CaMK-II genes10,11 whose protein products contain a sequence of MHRQE[pT287]VECLK in this region.10,11 This sequence is very similar to the phosphopeptide antigen used to create this rabbit polyclonal antibody (MHRQE[pT]VDCLK; Upstate/Millipore) and therefore it was not a complete surprise that this antibody cross-reacted with zebrafish CaMK-II. This laboratory showed that this antibody reacts with zebrafish CaMK-II in proportion to autophosphorylation and Ca2+/CaM-independent activity.12 Additional pan-specific CaMK-II antibodies have also been shown to cross-react with zebrafish CaMK-II.13
This antibody has been used to demonstrate that zebrafish CaMK-II is preferentially activated in cells on one side of the zebrafish Kupffer’s Vesicle (KV), the ciliated organ necessary for establishment of left/right asymmetry.12 This antibody was used to demonstrate that CaMK-II is transiently activated in four adjacent cells on the left side of the KV during the exact same developmental phase that organ positioning is determined.12 In addition to the Kupffer’s Vesicle (KV), autophosphorylated (P-T287) was also located in specific intracellular sites in other ciliated tissues including the kidney, neuromasts, and inner ear.12,13 In the zebrafish kidney, P-T287-CaMK-II is enriched along the apical border of ciliated ductal cells and within cloacal cilia where it influences their assembly.13 Finally, in the developing inner ear, P-T287-CaMK-II is intensely concentrated at the base of cilia and influences cell differentiation through the Delta-Notch signal pathway.14 In summary, the detection of activated CaMK-II has pinpointed sites of intracellular Ca2+ release and illuminated potential new signaling pathways.
These discoveries were completely dependent on developing a sensitive and accurate method to localize activated (P-T287-autophosphorylated) CaMK-II. The methods to fix and immunostain the zebrafish KV, kidney and inner ear are described. The limitations of this technique are also described. These techniques should be useful to any investigator who seeks to obtain high-resolution images in two fluorescent channels of not just phospho-epitopes, but any epitope, during early vertebrate development.
Protocol
Representative Results
Discussion
השיטה PFA / מתנול פותחה במעבדה זה במטרה העיקרית של אופטימיזציה של immunolocalization של epitope phospho-T 287 -CaMK-II במהלך ההתפתחות דג הזברה. שיטה זו מקומי בהצלחה P-CaMK-II במהלך היווצרות של מספר איברים ריסי כולל KV דג הזברה, 12 האוזן הפנימית 14 וכליות. 13 במיוחד בשלב KV, טכני?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
עבודה זו נתמכה על ידי מענק הקרן הלאומית למדע IOS-0,817,658.
Materials
| 1-phenyl-2-thiourea (PTU) | Sigma | P-7629 | 0.12% Stock solution. Dilute 1:40 in system water |
| Alexa488 anti-mouse IgG | Life Technologies | A11001 | Goat polyclonal, use at 1:500 |
| Alexa488 anti-rabbit IgG | Life Technologies | A11008 | Goat polyclonal, use at 1:500 |
| Alexa488 phalloidin | Life Technologies | A12379 | Preferentially binds to F-actin |
| Alexa568 anti-mouse IgG | Life Technologies | A11004 | Goat polyclonal, use at 1:500 |
| Alexa568 anti-rabbit IgG | Life Technologies | A11011 | Goat polyclonal, use at 1:500 |
| anti-acetylated a-tubulin | Sigma | T7451 | Mouse monoclonal, use at 1:500 |
| anti-phospho-T287 CaMK-II | EMD Millipore | 06-881 | Rabbit polyclonal, use at 1:20 |
| anti-total CaMK-II | BD Biosciences | 611292 | Mouse monoclonal, use at 1:20 |
| Ethanol | Fisher | S96857 | Lab grade, 95% denatured |
| Forceps | Fine Science Tools | 11252-20 | Dumont #5 |
| Glass coverslips | VWR | 16004-330 | #1 thickness |
| Glass microscope slides | Fisher | 12-550-15 | Standard glass slides |
| Methanol | Fisher | A411 | Store in freezer |
| Microcentrifuge tubes | VWR | 20170-038 | capped tubes, not sterile |
| Normal goat serum | Life Technologies | 16210-064 | Aliquot 1ml tubes, store in freezer |
| Paraformaldehyde | Sigma | P-6148 | Reagent grade, crystalline |
| Phosphate buffered saline (PBS) | Quality Biological | 119-069-131 | 10X stock solution or made in lab |
| Triton X-100 | Sigma | BP-151 | 10% solution in water, store at room temp |
| Tween-20 | Life Technologies | 85113 | 10% solution in water, store at room temp |
| Compound microscope | Nikon | E-600 | Mount on vibration-free table |
| C1 Plus two-laser scanning confocal | Nikon | C1 Plus | Run by EZ-C1 program, but upgrades use "Elements" |
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