Es sind Techniken beschrieben Phospho-Epitope in ganz Zebrafischembryonen zu Immunfärbung und dann leiten zweifarbige Leuchtstoff konfokalen Lokalisierung in zellularen Strukturen so klein wie primäre Zilien. Die Techniken zur Fixierung und Bildgebung können die Position und die Kinetik des Erscheinens oder Aktivierung spezifischer Proteine definieren.
Die schnelle Proliferation von Zellen, die gewebespezifische Expression von Genen und die Entstehung von Signalisierungsnetze charakterisieren frühen embryonalen Entwicklung aller Wirbeltiere. Die Kinetik und die Lage der Signale – auch innerhalb einzelner Zellen – in den sich entwickelnden Embryo ergänzt die Identifizierung wichtiger Entwicklungsgene. Immunfärbung Techniken beschrieben, die die Kinetik der intrazellulären und ganzen Tier Signalen in Strukturen so klein wie primäre Zilien definieren abgebildet wurden. Die Techniken zur Fixierung, Imaging und Bearbeitung von Bildern mit einem Laser-Scanning-konfokalen Mikroskops verwendet, kann in nur 36 Stunden abgeschlossen sein.
Zebrafisch (Danio rerio) ist eine wünschenswerte Organismus für Forscher, die versuchen, Studien in einem Wirbeltierarten durchzuführen, die erschwinglich und relevant für die menschliche Krankheit. Genetic Knockouts oder Niederschlägen muss durch den Verlust der tatsächlichen Proteinprodukt bestätigt. Eine solche Bestätigung von Proteinverlustwerden können unter Verwendung der hier beschriebenen Techniken erreicht. Clues der Signalwege kann auch durch Verwendung von Antikörpern entschlüsselt werden, der mit Proteinen reaktiv sind, die durch Phosphorylierung posttranslational modifiziert wurden. Die Erhaltung und die phosphorylierten Zustand eines Epitops Optimierung ist daher entscheidend für diese Bestimmung und wird von diesem Protokoll erreicht.
Diese Studie beschreibt Techniken Embryonen während der ersten 72 Stunden der Entwicklung und zu beheben eine Vielzahl von relevanten Epitope mit Zilien in das Vesikel (KV), die Nieren und das Innenohr des Kupffer co-lokalisieren. Diese Verfahren sind einfach, nicht Dissektion erforderlich und kann in einer relativ kurzen Zeitspanne abgeschlossen werden. Projizieren konfokale Bildstapel zu einem einzigen Bild ist ein nützliches Mittel, um diese Daten zu präsentieren.
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.
Die PFA / Methanol-Verfahren wurde in diesem Labor mit dem vorrangigen Ziel der Optimierung der Immun des Phospho-T 287 -CaMK-II-Epitop während Zebrabärblingentwicklung entwickelt. Diese Methode erfolgreich P-CaMK-II bei der Bildung von mehreren ciliated Organe, einschließlich Zebrafisch KV, 12 Innenohr 14 und Niere. 13 besonders am KV Stufe diese Technik notwendig war, lokalisiert. Der Erfolg dieses Verfahrens ist wahrscheinlich auf eine Kombination von a) Minimierung von …
The authors have nothing to disclose.
Diese Arbeit wurde von der National Science Foundation Grant IOS-0817658 unterstützt.
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" |