Technieken worden beschreven aan fosfo-epitopen geheel zebravis embryo immunostain en voert vervolgens twee kleuren fluorescerende confocale lokalisatie in cellulaire structuren zo klein als primaire cilia. De technieken voor de vaststelling en beeldvorming kan de locatie en de kinetiek van het uiterlijk of interactie met specifieke eiwitten te bepalen.
De snelle proliferatie van cellen, de weefsel-specifieke expressie van genen en het ontstaan van signaleringsnetwerken karakteriseren vroege embryonale ontwikkeling van gewervelde dieren. De kinetiek en de locatie van de signalen – zelfs binnen enkele cellen – in het zich ontwikkelende embryo een aanvulling op de identificatie van belangrijke ontwikkelingsstoornissen genen. Immunokleuring technieken beschreven die is aangetoond dat de kinetiek van intracellulaire en hele dieren signalen structuren zo klein als primaire cilia definiëren. De technieken voor het fixeren, beeldvorming en verwerking beelden met behulp van een laser-scanning confocale microscoop verbinding kan in slechts 36 uur worden voltooid.
De zebravis (Danio rerio) is een gewenst organisme voor onderzoekers die proberen om studies uit te voeren in een gewervelde soorten die betaalbaar is en voor de menselijke ziekten tegen te gaan. Genetische knockouts of knockdowns moet worden bevestigd door het verlies van de werkelijke eiwit product. Deze bevestiging van eiwitverlieskan worden bereikt met de hier beschreven technieken. Aanwijzingen in signaalroutes kunnen worden ontcijferd door gebruik van antilichamen die reageren met eiwitten die post-translationeel zijn gemodificeerd door fosforylering. Het behoud en het optimaliseren van de gefosforyleerde toestand van een epitoop is daarom van cruciaal belang voor deze vaststelling en wordt bereikt door dit protocol.
Deze studie beschrijft technieken voor embryo vast gedurende de eerste 72 uur van ontwikkeling en co-lokaliseren verschillende relevante epitopen met trilharen in Kupffer de blaasjes (KV), de nieren en het binnenoor. Deze technieken zijn eenvoudig, hoeft dissectie vereisen en kan in een relatief korte tijd worden voltooid. Projecteren image confocale stacks in een enkel beeld is een nuttig middel van de presentatie van deze gegevens.
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.
De PFA / methanol methode die in dit laboratorium met het hoofddoel van optimale immuunlokalisatie van de fosfo-T 287 -CaMK-II epitoop in zebravis ontwikkeling. Deze methode succesvol gelokaliseerde P-CaMK-II tijdens de vorming van verschillende trilharen organen waaronder de zebravis KV, 12 binnenoor 14 en nieren. 13 Met name in het stadium KV Deze techniek noodzakelijk was. Het succes van deze werkwijze is waarschijnlijk te wijten aan een combinatie van a) beperking van auto…
The authors have nothing to disclose.
Dit werk werd ondersteund door de National Science Foundation subsidie IOS-0817658.
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" |