Name: immunostaining phospho-epitopes in ciliated organs of whole mount zebrafish embryos
Uploaded: 2016-02-19T14:00:00.000+00:00
Duration: 8 min 42 s
Description: Watch this Scientific Journal Video about Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos at JoVE.com

Questo lavoro è stato sostenuto dalla sovvenzione della National Science Foundation IOS-0.817.658.

Le procedure di zebrafish in questo protocollo sono stati approvati dal Comitato Istituzionale Animal Care e Usa (IACUC) alla Virginia Commonwealth University. 1. Preparazione dei reagenti <ol><li> 4% PFA / PBS. Pesare 8 g di paraformaldeide (PFA) nella cappa. Sempre nella cappa, sciogliere il asciutta PFA in ~ 80 ml ​​di H 2 O distillata sotto agitazione e riscaldamento a 50 ° C. Mentre mescolando, aggiungere 3 - 10 gocce di fresca 1N NaOH fino PFA è completamente sciolto e la soluzione chiarisce. Togliere dal fuoco e aggiungere 100 ml di 2x tampone fosfato salino (PBS). Portare il volume a 200 ml con acqua distillata H 2 O. Raffreddare a RT e confermare pH di 7,4 prima dell&#39;uso. Conservare al buio a 4 ° C ma utilizzare entro una settimana. </li><li> Utilizzare 100% di metanolo senza supplementi. Utilizzare il 95% di etanolo senza supplementi. </li><li> Preparare fosfato-tamponata Tween (PBT). Supplemento PBS (PBS) con 0,1% di Tween-20. Aggiungi 0.5 ml di Tween-20 soluzione madre 20% a 100 ml 1x PBS. Conservare a temperatura ambiente. </li><li> Preparare fosfato-tamponata Triton-X (PBTx). Supplemento PBS con 0,1% Triton X-100. Aggiungere 0,5 ml di 20% Triton X-100 magazzino a 100 ml di PBS. Conservare a temperatura ambiente. </li><li> Preparare 10% NGS / PBTx. blocchi normali siero di capra (NGS) non specifici siti di legame. NGS Conservare in aliquote a -20 ° C. Per utilizzare, aggiungere 1 ml a 9 ml PBTx. </li><li> Preparare 50% glicerolo / PBS. Mescolare parti uguali di fondo 2x PBS e 100% glicerolo. Conservare a temperatura ambiente. </li></ol> 2. Embryo Fixation <ol><li> . (Es., Β-actina: CAAX-GFP) Allevamento ottenere wild type (AB e WIK) o transgenici embrioni attraverso accoppiamenti naturali. Se lo si desidera, iniettare embrioni con costrutti o morpholinos, come descritto. 15,16 </li><li> Sollevare embrioni. Raccogliere embrioni e incubare a 28,5 ° C in presenza di 0,003% 1-fenil-2-tiourea (PTU) per bloccare la pigmentazione come descritto. 17 </su<html> p&gt; </li><li> Fissare. Quando gli embrioni hanno raggiunto lo stadio di sviluppo desiderato, 18, &#160; anestetizzare gli embrioni utilizzando MESAB, rimuovere l&#39;acqua sistema quanto più possibile e aggiungere fresca 4% PFA / PBS per 3 - 4 ore a RT. NOTA: A volte meno di 20 ore dopo la fecondazione (HPF), fissare prima dechorionation. A volte dopo 20 HPF, dechorionate prima della fissazione con due coppie di pinze punta fine sotto un microscopio da dissezione. </li><li> Cambiare soluzioni. Trasferire gli embrioni utilizzando una vasta pipetta foro, come descritto. 17 Variazione soluzioni in 1,5 ml con tappo provette da microcentrifuga, semplicemente permettendo embrioni di stabilirsi per gravità senza centrifugazione. </li><li> Post-fissativo Dopo 3 -. 4 ore, rimuovere PFA / PBS, sostituire con il 100% di metanolo e conservare a -20 ° C per almeno 48 ore. NOTA: per la massima immunoreattività P-CaMK-II, limitare il tempo totale di memorizzazione metanolo per una settimana. </li></ol>&quot;&gt; 3. Immunocolorazione intero embrioni <ol><li> Posizionare un minimo di 10 embrioni per ogni condizione sperimentale in innevate provetta da 1,5 ml microcentrifuga, ed etichettarli. </li><li> Lasciare gli embrioni di stabilirsi. Rimuovere e scartare metanolo e reidratare con lavaggi progressive contenenti 0,5 ml di concentrazioni decrescenti di etanolo, come indicato nel passaggio successivo. Ogni passo è a 5 minuti di lavaggio con dondolo. </li><li> Progressivamente reidratare con questi 5 soluzioni: 66% etanolo / 33% PBTx, 33% etanolo / 66% PBTx, 100% PBTx, 100% PBTx (questa è la prima ripetizione di PBTx), 100% PBTx (questa è la seconda ripetizione PBTx). </li><li> Rimuovere ultimo lavaggio PBTx. Aggiungere 0,5 ml di 10% NGS / PBTx. Incubare per almeno 1 ora a temperatura ambiente con dolce dondolio e poi rimuovere ed eliminare soluzione. </li><li> Preparare la soluzione anticorpo primario diluendo nel 10% NGS / PBTx. Se la co-immunostaining, usare degli anticorpi più alta affinità per primo. Per questo studio, incubare con il mouse anti-acetilata anticorpo monoclonale tubulina diluito 1: 500. per example, se ci sono 10 campioni, combinare 6 ml di soluzione madre di anticorpi con 3 ml di 10% NGS / PBTx quindi distribuire 0,3 ml di questa in ogni provetta. </li><li> Aggiungere 0,2 - 0,5 ml di anticorpo primario diluito in ogni provetta per immergere tutti gli embrioni. Incubare con dolce dondolio O / N a temperatura ambiente. </li><li> Al mattino, rimuovere ed eliminare soluzione di anticorpi. Aggiungere 0,5 ml 2% NGS / PBTx per lavare via anticorpo primario in eccesso. Agitare delicatamente per 5 min. Rimuovere e scartare la soluzione. Ripetere due volte. </li><li> Dim plafoniere e diluire l&#39;anticorpo secondario fluorescente coniugato adeguato nel 10% NGS / PBTx in modo che ogni condizione contiene almeno 0,5 ml. Con il topo anti-acetilata anticorpo primario tubulina, utilizzare il verde-colorante fluorescente capra anti-IgG di topo ad una diluizione 1: 500. </li><li> Incubare anticorpo secondario per 4 ore con leggera oscillazione a RT al buio. D&#39;ora in poi, il processo di campioni sotto luci soffuse e incubare in un contenitore scuro o avvolgendo in un foglio di alluminio. </li><li>Al termine dell&#39;incubazione, rimuovere e scartare la soluzione di anticorpo secondario. Aggiungere 0,5 ml 2% NGS / PBTx per lavare. Agitare delicatamente per 5 min. Rimuovere e scartare la soluzione. Ripetere due volte. </li><li> Se la co-immunostaining, ottenere il secondo anticorpo primario da una specie diversa rispetto al primo anticorpo primario. In questi studi, seguire l&#39;anticorpo CaMK-II di coniglio anti-fosfo dal colorante coniugato capra anti-coniglio anticorpo secondario rosso fluorescente IgG. </li><li> Diluire coniglio anti-fosfo-CaMK II anticorpi 1:20. Per ogni tubo di embrioni, la sospensione in 0,3 ml di 10% NGS / PBTx, quindi aggiungere 15 ml di soluzione madre di anticorpi. </li><li> Assicurarsi che gli embrioni sono immersi e le luci si abbassano. Incubare con dolce O dondolo / N a RT al buio. </li><li> La mattina sotto le luci soffuse, rimuovere ed eliminare soluzione di anticorpi. Aggiungere 0,5 ml 2% NGS / PBTx. Agitare delicatamente per 5 min. Rimuovere e scartare la soluzione. Ripetere due volte. </li><li> Mantenere plafoniere fioca e abbastanza del appropriata fluorescenza conju diluiregated anticorpo secondario in modo che ogni condizione contiene almeno 0,5 ml. In questo studio, diluire il colorante coniugato capra anti-IgG di coniglio anticorpi rosso fluorescente secondaria (canale rosso) 1: 500 nel 10% NGS / PBTx. </li><li> Incubare secondo anticorpo secondario per 4 ore con leggera oscillazione a RT al buio. </li><li> Alla fine di questa incubazione e sotto le luci soffuse, rimuovere ed eliminare la soluzione di anticorpo secondario. Aggiungere 0,5 ml PBTx. Agitare delicatamente per 5 min. Rimuovere e scartare la soluzione. Ripetere due volte. Conservare in entrambi PBTx o il 50% glicerolo / PBS a seconda della procedura di imaging. </li></ol> 4. confocale Imaging and Processing <ol><li> Monte embrioni per l&#39;imaging. Posizionare 1 - 5 embrioni su un vetrino. Creare una camera tra il vetrino principale e la diapositiva utilizzando frammenti coprioggetto su ciascun lato della camera. Tipicamente, quattro # 1 coprioggetto sono usati per fare queste pile distanziatori senza incollarsi. NOTA: Nessun mezzo di montaggio è necessario. </li><li> Utilizzare una immersione in olio 100Xobiettivo di portare singolo embrione a fuoco con luce trasmessa. Spegnere la luce. Accendere microscopio confocale. Garantire che i laser appropriate (verde e / o rosso) sono accesi e accessary messa a fuoco remota è impegnata. </li><li> Attivare il programma confocale. Nella &quot;barra Acquisisci&quot;, selezionare l&#39;obiettivo corretto. Nella barra &quot;XY di base&quot;, impostare le dimensioni dell&#39;immagine a 1.024 facendo clic sul pulsante 1.024. </li><li> Nella &quot;Laser e Detector&quot; bar, clicca con il 488 box rosso e il verde 568 casella per attivare il laser e rivelatore per ciascun canale. Impostare pinhole a medio e regolare il guadagno nella barra &quot;Gain&quot; di ciascun canale da visualizzare. </li><li> Nella barra &quot;Acquisire Impostazioni&quot;, cliccare su &quot;Live&quot; per iniziare l&#39;acquisizione di immagini. Nella barra &quot;View Settings&quot;, deselezionare la casella &quot;Forza integrale Zoom&quot; al centro nella finestra &quot;Live&quot;. </li><li> Nella barra &quot;Acquisisci impostazioni&quot;, nella scheda &quot;Z&quot;, scorrere i livelli dell&#39;immagine. Dopo aver selezionato il number di sezioni ottiche incorporare nella immagine, passare a un certo punto nel &quot;centro&quot; del piano z. </li><li> Nella scheda &quot;Z&quot;, fare clic sulla piccola scatola rossa &quot;di riferimento&quot;. Questo azzererà la RFA nel punto prescelto come &quot;centro&quot;. Nella casella &quot;Step Size&quot;, selezionare lo spessore degli strati (tra 0,25 micron e 2,0 micron è l&#39;ideale). Prestare attenzione alla casella &quot;Formato File&quot; e cercare di limitare a 1 GB. NOTA: in genere, fino a 40 sezioni ottiche di 0,5 - 1,0 micron si ottengono, ma il numero di sezioni può essere determinato empiricamente. </li><li> Per trovare l&#39;estremo superiore dell&#39;immagine, fare clic sul cerchio accanto a &quot;Top&quot; e passare attraverso gli strati z. Ora clicca il cerchio accanto a &quot;basso&quot;, il computer avrà l&#39;immagine di nuovo al livello come il centro. Ancora spostarsi attraverso gli strati di trovare l&#39;estremo inferiore dell&#39;immagine. </li><li> Clicca su &quot;vivere&quot; ancora una volta nei &quot;Acquisisci Impostazioni&quot; di dialogo per fermare il laseracquisizione. In questo pannello, selezionare le caselle rosse etichettati media e Z-stack. Queste scatole diventa verde. </li><li> Nella casella &quot;Acquisire Impostazioni&quot;, fai clic su &quot;Single&quot; per acquisire l&#39;intera serie di immagini. È possibile monitorare lo stato di avanzamento, come la scansione in entrambi i canali e attraverso l&#39;intero z-stack. </li><li> Per risparmiare, fare clic sulla finestra del volume e fare clic su &quot;Salva con nome&quot; e il nome del file. Salvare come file &quot;.ids&quot;. Per volume di rendere il file mentre lo z-stack è ancora aperto, selezionare i dati menu a discesa e clicca su &quot;il volume di rendering&quot;. Salvare il file reso come un file TIFF. Questa è l&#39;immagine proiettata che sono presenti in questa pubblicazione. </li></ol>

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Gli autori dichiarano di non avere interessi finanziari concorrenti.

Il metodo / metanolo PFA è stato sviluppato in questo laboratorio con l&#39;obiettivo primario di ottimizzare la immunolocalizzazione del fosfo-T 287 -CaMK-II epitopi durante lo sviluppo zebrafish. Questo metodo localizzato successo P-CaMK-II durante la formazione di diversi organi ciliati compreso il KV zebrafish, 12 orecchio interno 14 e reni. 13 In particolare nella fase KV, questa tecnica era necessario. Il successo di questo metodo è probabilmente dovuto ad una combinaz...

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.

<table><tbody><tr><td>1-phenyl-2-thiourea (PTU)</td><td>Sigma</td><td>P-7629</td><td>0.12% Stock solution. Dilute 1:40 in system water</td></tr><tr><td>Alexa488 anti-mouse IgG</td><td>Life Technologies</td><td>A11001</td><td>Goat polyclonal, use at 1:500</td></tr><tr><td>Alexa488 anti-rabbit IgG</td><td>Life Technologies</td><td>A11008</td><td>Goat polyclonal, use at 1:500</td></tr><tr><td>Alexa488 phalloidin</td><td>Life Technologies</td><td>A12379</td><td>Preferentially binds to F-actin</td></tr><tr><td>Alexa568 anti-mouse IgG</td><td>Life Technologies</td><td>A11004</td><td>Goat polyclonal, use at 1:500</td></tr><tr><td>Alexa568 anti-rabbit IgG</td><td>Life Technologies</td><td>A11011</td><td>Goat polyclonal, use at 1:500</td></tr><tr><td>anti-acetylated &amp;#945;-tubulin</td><td>Sigma</td><td>T7451</td><td>Mouse monoclonal, use at 1:500</td></tr><tr><td>anti-phospho-T&lt;sup&gt;287&lt;/sup&gt; CaMK-II</td><td>EMD Millipore</td><td>06-881</td><td>Rabbit polyclonal, use at 1:20</td></tr><tr><td>anti-total CaMK-II</td><td>BD Biosciences</td><td>611292</td><td>Mouse monoclonal, use at 1:20</td></tr><tr><td>Ethanol</td><td>Fisher</td><td>S96857</td><td>Lab grade, 95% denatured</td></tr><tr><td>Forceps</td><td>Fine Science Tools</td><td>11252-20</td><td>Dumont #5</td></tr><tr><td>Glass coverslips</td><td>VWR</td><td>16004-330</td><td>#1&amp;nbsp; thickness</td></tr><tr><td>Glass microscope slides</td><td>Fisher</td><td>12-550-15</td><td>Standard glass slides</td></tr><tr><td>Methanol</td><td>Fisher</td><td>A411</td><td>Store in freezer</td></tr><tr><td>Microcentrifuge tubes</td><td>VWR</td><td>20170-038</td><td>capped tubes, not sterile</td></tr><tr><td>Normal goat serum</td><td>Life Technologies</td><td>16210-064</td><td>Aliquot 1 ml tubes, store in freezer</td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>P-6148</td><td>Reagent grade, crystalline</td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Quality Biological</td><td>119-069-131</td><td>10x stock solution or made in lab</td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>BP-151</td><td>10% solution in water, store at RT</td></tr><tr><td>Tween-20</td><td>Life Technologies</td><td>85113</td><td>10% solution in water, store at RT</td></tr><tr><td>Compound microscope</td><td>Nikon</td><td>E-600</td><td>Mount on vibration-free table</td></tr><tr><td>C1 Plus two-laser scanning confocal</td><td>Nikon</td><td>C1 Plus</td><td>Run by EZ-C1 program, but upgrades use &quot;Elements&quot;</td></tr></tbody></table>

immunostaining phospho-epitopes in ciliated organs of whole mount zebrafish embryos

La rapida proliferazione delle cellule, l&#39;espressione tessuto-specifica di geni e la nascita di reti di segnalazione caratterizzano sviluppo embrionale precoce di tutti i vertebrati. La cinetica e la posizione dei segnali - anche all&#39;interno di singole cellule - negli embrioni integra l&#39;identificazione di importanti geni dello sviluppo. tecniche di immunoistochimica sono descritte che hanno dimostrato di definire la cinetica di segnali intracellulari animali e intere in strutture piccole come cilia primaria. Le tecniche per il fissaggio, di imaging e di elaborazione delle immagini utilizzando un microscopio a scansione laser confocale composto può essere completato in soli 36 hr. Zebrafish (Danio rerio) è un organismo desiderabile per gli investigatori che cercano di condurre studi in una specie di vertebrati che è conveniente e rilevanti per le malattie umane. ko genetica o abbattimenti devono essere confermati dalla perdita del prodotto proteico reale. Tale conferma della perdita di proteinepuò essere realizzato utilizzando le tecniche descritte qui. Gli indizi in vie di segnalazione possono essere decifrati utilizzando anticorpi che sono reattivi con le proteine ​​che sono stati post-traduzionali modificati dalla fosforilazione. Preservare e ottimizzare lo stato fosforilato di un epitopo è quindi fondamentale per questa determinazione e si realizza con questo protocollo. Questo studio descrive tecniche per risolvere embrioni durante il primo 72 hr di sviluppo e co-localizzare una varietà di epitopi rilevanti con cilia nella zona di Kupffer Vesicle (KV), il rene e l&#39;orecchio interno. Queste tecniche sono semplici, non richiedono la dissezione e può essere completato in un periodo relativamente breve di tempo. Proiezione pile di immagini confocale in una singola immagine è un mezzo utile per la presentazione di questi dati.

Condizioni ottimali per la visualizzazione di fosfo-epitopi I metodi che descrivono la immunolocalizzazione di epitopi della proteina negli embrioni di zebrafish sono state relativamente scarse rispetto a quelle per la localizzazione mRNA tramite ibridazione in situ. Fissativi utilizzati nella localizzazione epitopi di proteine ​​nelle cellule ciliate di embrioni di zebrafish hanno incluso 4% PFA ...

Watch this Scientific Journal Video about Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos at JoVE.com

Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos

Le tecniche sono descritte a immunostain fosfo-epitopi in interi embrioni di zebrafish e quindi condurre a due colori confocale a fluorescenza localizzazione in strutture cellulari piccoli come ciglia primarie. Le tecniche di fissaggio e di imaging possono definire la posizione e cinetica di comparsa o l&#39;attivazione di specifiche proteine.

Immunocolorazione fosfo-epitopi in Ciliated Organi di monte intero embrioni di zebrafish

The rapid proliferation of cells, the tissue-specific expression of genes and the emergence of signaling networks characterize early embryonic development ...

immunostaining-phospho-epitopes-ciliated-organs-whole-mount-zebrafish

Research

JoVE Journal

Chemistry

8.0K Views.  Virginia Commonwealth University.  Le tecniche sono descritte a immunostain fosfo-epitopi in interi embrioni di zebrafish e quindi condurre a due colori confocale a fluorescenza localizzazione in strutture cellulari piccoli come ciglia primarie. Le tecniche di fissaggio e di imaging possono definire la posizione e cinetica di comparsa o l'attivazione di specifiche proteine. 

Video: Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos

High Precision Zinc Isotopic Measurements Applied to Mouse Organs

We present a procedure to measure with high precision zinc isotope ratios in mouse organs. Zinc is composed of 5 stable isotopes (64Zn, 66Zn, 67Zn, 68Zn and 70Zn) which are naturally fractionated between mouse organs. We first show how to dissolve the different organs in order to free the Zn atoms; this step is realized by a mixture of HNO3 and H2O2. We then purify the zinc atoms from all the other elements, in particular from isobaric interferences (e.g., Ni), by anion-exchange chromatography in a dilute HBr/HNO3 medium. These first two steps are performed in a clean laboratory using high purity chemicals. Finally, the isotope ratios are measured by using a multi-collector inductively-coupled-plasma mass-spectrometer, in low resolution. The samples are injected using a spray chamber and the isotopic fractionation induced by the mass-spectrometer is corrected by comparing the ratio of the samples to the ratio of a standard (standard bracketing technique).&#160;This full typical&#160;procedure produces an isotope ratio with a 50 ppm (2 s.d.) reproducibility.

We present the technique to measure with high precision zinc isotope ratios in mouse organs.

Alte le misure di precisione di zinco isotopiche applicate a mouse Organi

We present a procedure to measure with high precision zinc isotope ratios in mouse organs. Zinc is composed of 5 stable isotopes (64Zn, 66Zn, 67Zn, 68Zn ...

Ricerca

Chimica

Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular identity and function. Each of the seven PtdInsPs varies in their distribution and abundance, which are tightly regulated by specific kinases and phosphatases. The abundance of PtdInsPs can change abruptly in response to various signaling events or disturbance of the regulatory machinery. To understand how these events lead to changes in the amount of PtdInsPs and their resulting impact, it is important to quantify PtdInsP levels before and after a signaling event or between control and abnormal conditions. However, due to their low abundance and similarity, quantifying the relative amounts of each PtdInsP can be challenging. This article describes a method for quantifying PtdInsP levels by metabolically labeling cells with 3H-myo-inositol, which is incorporated into PtdInsPs. Phospholipids are then precipitated and deacylated. The resulting soluble 3H-glycero-inositides are further extracted, separated by high-performance liquid chromatography (HPLC), and detected by flow scintillation. The labeling and processing of yeast samples is described in detail, as well as the instrumental setup for the HPLC and flow scintillator. Despite losing structural information regarding acyl chain content, this method is sensitive and can be optimized to concurrently quantify all seven PtdInsPs in cells.

Phosphoinositides are signaling lipids whose relative abundance rapidly changes in response to various stimuli. This article describes a method to measure the abundance of phosphoinositides by metabolically labeling cells with 3H-myo-inositol, followed by extraction and deacylation. Extracted glycero-inositides are then separated by high-performance liquid chromatography and quantified by flow scintillation.

Radiomarcatura e quantificazione dei livelli cellulari di fosfoinositidi da alte prestazioni cromatografia liquida accoppiata flusso scintillazione

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular ...

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and optical transparency. In particular, the zebrafish embryo has become an important organism to study vertebrate kidney organogenesis as well as multiciliated cell (MCC) development. To visualize MCCs in the embryonic zebrafish kidney, we have developed a combined protocol of whole-mount fluorescent in situ hybridization (FISH) and whole mount immunofluorescence (IF) that enables high resolution imaging. This manuscript describes our technique for co-localizing RNA transcripts and protein as a tool to better understand the regulation of developmental programs through the expression of various lineage factors.

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

Cilia development is vital to proper organogenesis. This protocol describes an optimized method to label and visualize ciliated cells of the zebrafish.

Visualizzazione di cellule Multiciliated in Zebrafish attraverso un protocollo combinato di tutta Monte fluorescente ibridazione In Situ e immunofluorescenza

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and ...

Biologia dello sviluppo

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red 1,2. This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) 3. The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy 3,4. One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein 5. When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. We include a propidium iodide nuclear counter-stain to highlight tissue organization.

Zebrafish intero monte ad alta risoluzione doppia fluorescente In Situ Ibridazione

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

Biologia

Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. Although many immunohistochemistry protocols have been optimized for mammalian tissue and tissue sections, these protocols often require modification and optimization for non-mammalian model organisms. Zebrafish are increasingly used as a model system in basic, biomedical, and translational research to investigate the molecular, genetic, and cell biological mechanisms of developmental processes. Zebrafish offer many advantages as a model system but also require modified techniques for optimal protein detection. Here, we provide our protocol for whole-mount fluorescence immunohistochemistry in zebrafish embryos and larvae. This protocol additionally describes several different mounting strategies that can be employed and an overview of the advantages and disadvantages each strategy provides. We also describe modifications to this protocol to allow detection of chromogenic substrates in whole mount tissue and fluorescence detection in sectioned larval tissue. This protocol is broadly applicable to the study of many developmental stages and embryonic structures.

Here, we present a protocol for fluorescent antibody-mediated detection of proteins in whole preparations of zebrafish embryos and larvae.

Intero Monte Immunohistochimica in Embrioni e Larve di pesce zebra

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. ...

Single Cell Fate Mapping in Zebrafish

The ability to differentially label single cells has important implications in developmental biology. For instance, determining how hematopoietic, lymphatic, and blood vessel lineages arise in developing embryos requires fate mapping and lineage tracing of undifferentiated precursor cells. Recently, photoactivatable proteins which include: Eos1, 2, PAmCherry3, Kaede4-7, pKindling8, and KikGR9, 10 have received wide interest as cell tracing probes. The fluorescence spectrum of these photosensitive proteins can be easily converted with UV excitation, allowing a population of cells to be distinguished from adjacent ones. However, the photoefficiency of the activated protein may limit long-term cell tracking11. As an alternative to photoactivatable proteins, caged fluorescein-dextran has been widely used in embryo model systems7, 12-14. Traditionally, to uncage fluorescein-dextran, UV excitation from a fluorescence lamp house or a single photon UV laser has been used; however, such sources limit the spatial resolution of photoactivation. Here we report a protocol to fate map, lineage trace, and detect single labeled cells. Single cells in embryos injected with caged fluorescein-dextran are photoactivated with near-infrared laser pulses produced from a titanium sapphire femtosecond laser. This laser is customary in all two-photon confocal microscopes such as the LSM 510 META NLO microscope used in this paper. Since biological tissue is transparent to near-infrared irradiation15, the laser pulses can be focused deep within the embryo without uncaging cells above or below the selected focal plane. Therefore, non-linear two-photon absorption is induced only at the geometric focus to uncage fluorescein-dextran in a single cell. To detect the cell containing uncaged fluorescein-dextran, we describe a simple immunohistochemistry protocol16 to rapidly visualize the activated cell. The activation and detection protocol presented in this paper is versatile and can be applied to any model system. 
Note: The reagents used in this protocol can be found in the table appended at the end of the article.

A method is described to photoactivate single cells containing a caged fluorescent protein using two-photon absorption from a Ti:Sapphire femtosecond laser oscillator. To fate map the photoactivated cell, immunohistochemistry is used. This technique can be applied to any cell type.

Singolo Destino Mapping di celle in Zebrafish

The ability to differentially label single cells has important implications in developmental biology. For instance, determining how hematopoietic, ...

Immunostaining of Dissected Zebrafish Embryonic Heart 

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology and genetics 1,2. About 100-200 embryos are readily available every week from a single pair of adult fish. The transparent embryos that develop ex utero make them ideal for assessing cardiac defects 3. The expression of any gene can be manipulated via morpholino technology or RNA injection 4. Moreover, forward genetic screens have already generated a list of mutants that affect different perspectives of cardiogenesis 5. 
Whole mount immunostaining is an important technique in this animal model to reveal the expression pattern of the targeted protein to a particular tissue 6. However, high resolution images that can reveal cellular or subcellular structures have been difficult, mainly due to the physical location of the heart and the poor penetration of the antibodies. 
 Here, we present a method to address these bottlenecks by dissecting heart first and then conducting the staining process on the surface of a microscope slide. To prevent the loss of small heart samples and to facilitate solution handling, we restricted the heart samples within a circle on the surface of the microscope slides drawn by an immEdge pen. After the staining, the fluorescence signals can be directly observed by a compound microscope. 
Our new method significantly improves the penetration for antibodies, since a heart from an embryonic fish only consists of few cell layers. High quality images from intact hearts can be obtained within a much reduced procession time for zebrafish embryos aged from day 2 to day 6. Our method can be potentially extended to stain other organs dissected from either zebrafish or other small animals.

Immunostaining of Dissected Zebrafish Embryonic Heart

A rapid way to conduct immunostaining of zebrafish embryonic heart is described. Compared to the whole mount immunostaining approach, this method dramatically increases the penetration of the antibodies, which allows obtaining high resolution images that reveal cellular/subcellular structures in the heart within a much reduced processing time.

Immunocolorazione di Dissected Cuore Zebrafish embrionali

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology ...

An Efficient Protocol to Assess ERK Activity Modulation in Early Zebrafish Noonan Syndrome Models via Live FRET Microscopy and Immunofluorescence

RASopathies are genetic syndromes caused by ERK hyperactivation and resulting in multisystemic diseases that can also lead to cancer predisposition. Despite a broad genetic heterogeneity, germline gain-of-function mutations in key regulators of the RAS-MAPK pathway underlie the majority of the cases, and, thanks to advanced sequencing techniques, potentially pathogenic variants affecting the RAS-MAPK pathway continue to be identified. Functional validation of the pathogenicity of these variants, essential for accurate diagnosis, requires fast and reliable protocols, preferably in vivo. Given the scarcity of effective treatments in early childhood, such protocols, especially if scalable in cost-effective animal models, can be instrumental in offering a preclinical ground for drug repositioning/repurposing. 
Here we describe step-by-step the protocol for rapid generation of transient RASopathy models in zebrafish embryos and direct inspection of live disease-associated ERK activity changes occurring already during gastrulation through real-time multispectral F&#246;rster resonance energy transfer (FRET) imaging. The protocol uses a transgenic ERK reporter recently established and integrated with the hardware of commercial microscopes. We provide an example application for Noonan syndrome (NS) zebrafish models obtained by expression of the Shp2D61G. We describe a straightforward method that enables registration of ERK signal change in the NS fish model before and after pharmacological signal modulation by available low-dose MEK inhibitors. We detail how to generate, retrieve, and assess ratiometric FRET signals from multispectral acquisitions before and after treatment and how to cross-validate the results via classical immunofluorescence on whole embryos at early stages. We then describe how, via examining standard morphometric parameters, to query late changes in embryo shape, indicative of a resulting impairment of gastrulation, in the same embryos whose ERK activity is assessed by live FRET at 6 h post fertilization.

RASopathies are multisystem genetic syndromes caused by RAS-MAPK pathway hyperactivation. Potentially pathogenic variants awaiting validation emerge continuously while poor preclinical evidence limits therapy. Here, we describe our in vivo protocol to test and cross-validate RASopathy-associated ERK activation levels and its pharmacological modulation during embryogenesis by live FRET imaging in Teen-reporter zebrafish.

Un protocollo efficiente per valutare la modulazione dell'attività di ERK nei primi modelli di sindrome di Noonan del pesce zebra tramite microscopia FRET dal vivo e immunofluorescenza

RASopathies are genetic syndromes caused by ERK hyperactivation and resulting in multisystemic diseases that can also lead to cancer predisposition. ...

Biochimica

In Situ Hybridization Combined with Immunohistochemistry in Cryosectioned Zebrafish Embryos

As a vertebrate, the zebrafish has been widely used in biological studies. Zebrafish and humans share high genetic homology, which allows its use as a model for human diseases. Gene function study is based on the detection of gene expression patterns. Although immunohistochemistry offers a powerful way to assay protein expression, the limited number of commercially available antibodies in zebrafish restricts the application of costaining. In situ hybridization is widely used in zebrafish embryos to detect mRNA expression. This protocol describes how to obtain images by combining in situ hybridization and immunohistochemistry for zebrafish embryo sections. In situ hybridization was performed prior to cryosectioning, followed by antibody staining. Immunohistochemistry and the imaging of a single cryosection were performed after in situ hybridization. The protocol is helpful to unravel the expression pattern of two genes, first by in situ transcript detection and then by immunohistochemistry against a protein in the same section.

In Situ Hybridization Combined with Immunohistochemistry in Cryosectioned Zebrafish Embryos

This protocol describes how to obtain images by combining in situ hybridization and immunohistochemistry of zebrafish embryonic sections. In situ hybridization was performed prior to cryosectioning, followed by antibody staining. It is useful to detect the expression patterns of two genes in zebrafish if there is a paucity of antibodies.

Sul posto Ibridazione combinata con immunoistochimica in embrioni di zebrafish criosezionati

As a vertebrate, the zebrafish has been widely used in biological studies. Zebrafish and humans share high genetic homology, which allows its use as a ...

Neuroscienze

Sequential Immunofluorescence and Immunohistochemistry on Cryosectioned Zebrafish Embryos

Investigation of intercellular interactions often requires discrete labeling of specific cell populations and precise protein localization. The zebrafish embryo is an excellent tool for examining such interactions with an in vivo model. Whole-mount immunohistochemical and immunofluorescence assays are frequently applied in zebrafish embryos to assess protein expression. However, it can be difficult to achieve accurate mapping of co-localized proteins in three-dimensional space. In addition, some studies may require the use of two antibodies that are not compatible with the same technique (e.g., antibody 1 is only suitable for immunohistochemistry and antibody 2 is only suitable for immunofluorescence). The purpose of the method described herein is to perform sequential immunofluorescence and/or immunohistochemistry on individual cryosections derived from early-stage zebrafish embryos. Here we describe the use of sequential rounds of immunofluorescence, imaging, immunohistochemistry, imaging for a single cryosection in order to achieve precise identification of protein expression at the single-cell level. This methodology is suitable for any study in early-stage zebrafish embryos that requires accurate identification of multiple protein targets in individual cells.

This protocol demonstrates sequential immunofluorescence and immunohistochemistry on cryosections from early-stage zebrafish embryos enabling precise colocalization analyses in specific cell populations.

Immunofluorescenza sequenziale e immunosofochimica su embrioni di pesce zebra criosezione

Investigation of intercellular interactions often requires discrete labeling of specific cell populations and precise protein localization. The zebrafish ...