Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos
PRÉPARATION DE L'INSTRUCTEUR
concepts
PROTOCOLE ETUDIANT
The zebrafish procedures in this protocol have been approved by the Institutional Animal Care and Use Committee (IACUC) at Virginia Commonwealth University.
1. Preparation of Reagents
- 4% PFA/PBS. Weigh 8 g of paraformaldehyde (PFA) in the fume hood. While still in the fume hood, dissolve the dry PFA in ~80 ml distilled H2O with stirring and heating to 50 °C. While stirring, add 3 – 10 drops of fresh 1N NaOH until PFA is completely dissolved and solution clarifies. Remove from heat and add 100 ml 2x phosphate buffered saline (PBS). Bring volume to 200 ml with distilled H2O. Cool to RT and confirm pH of 7.4 before use. Store in the dark at 4 °C but use within one week.
- Use 100% methanol without any supplements. Use 95% ethanol without any supplements.
- Prepare Phosphate-buffered Tween (PBT). Supplement Phosphate-buffered saline (PBS) with 0.1% Tween-20. Add 0.5 ml of a 20% Tween-20 stock solution to 100 ml 1x PBS. Store at RT.
- Prepare Phosphate-buffered Triton-X (PBTx). Supplement PBS with 0.1% Triton X-100. Add 0.5 ml of a 20% Triton X-100 stock to 100 ml of PBS. Store at RT.
- Prepare 10% NGS/PBTx. Normal goat serum (NGS) blocks non-specific binding sites. Store NGS in aliquots at -20 °C. To use, add 1 ml to 9 ml PBTx.
- Prepare 50% glycerol/PBS. Mix thoroughly equal parts of 2x PBS and 100% glycerol. Store at RT.
2. Embryo Fixation
- Breeding. Obtain wild type (AB and WIK) or transgenic (e.g., β-actin:CAAX-GFP) embryos through natural matings. If desired, inject embryos with constructs or morpholinos, as described.15,16
- Raise embryos. Collect embryos and incubate at 28.5 °C in the presence of 0.003% 1-phenyl-2-thiourea (PTU) to block pigmentation as described.17
- Fix. When embryos have reached the desired developmental stage,18, anesthetize the embryos using MESAB, remove as much system water as possible and add fresh 4% PFA/PBS for 3 – 4 hr at RT. NOTE: At times less than 20 hr post fertilization (hpf), fix prior to dechorionation. At times after 20 hpf, dechorionate prior to fixation using two pairs of fine point forceps under a dissecting microscope.
- Changing solutions. Transfer embryos using a wide bore pipet, as described.17 Change solutions in 1.5 ml capped microcentrifuge tubes, simply by allowing embryos to settle by gravity without centrifugation.
- Post-fixative. After 3 – 4 hr, remove PFA/PBS, replace with 100% methanol and store at -20 °C for at least 48 hr. NOTE: For maximal P-CaMK-II immunoreactivity, limit total methanol storage time to one week.
3. Immunostaining Whole Embryos
- Place a minimum of 10 embryos for each experimental condition in capped 1.5 ml microcentrifuge tube, and label them.
- Allow the embryos to settle. Remove and discard methanol and rehydrate with progressive washes containing 0.5 ml of decreasing concentrations of ethanol, as indicated in the next step. Each step is a 5 min wash with rocking.
- Progressively rehydrate with these 5 solutions: 66% ethanol/33% PBTx, 33% ethanol/66% PBTx, 100% PBTx, 100% PBTx (this is the first repeat of PBTx), 100% PBTx (this is the second repeat of PBTx).
- Remove last PBTx wash. Add 0.5 ml 10% NGS/PBTx. Incubate for at least 1 hr at RT with gentle rocking and then remove and discard solution.
- Prepare primary antibody solution by diluting in 10% NGS/PBTx. If co-immunostaining, use higher affinity antibody first. For this study, incubate with the mouse anti-acetylated tubulin monoclonal antibody diluted 1:500. For example, if there are 10 samples, combine 6 μl of the stock antibody solution with 3 ml of 10% NGS/PBTx and then distribute 0.3 ml of this into each tube.
- Add 0.2 – 0.5 ml of the diluted primary antibody to each tube to immerse all embryos. Incubate with gentle rocking O/N at RT.
- In the morning, remove and discard antibody solution. Add 0.5 ml 2% NGS/PBTx to wash away excess primary antibody. Gently rock for 5 min. Remove and discard solution. Repeat twice.
- Dim overhead lights and dilute the appropriate fluorescently-conjugated secondary antibody in 10% NGS/PBTx so that each condition contains at least 0.5 ml. With the mouse anti-acetylated tubulin primary antibody, use the green-fluorescent dye goat anti-mouse IgG at a 1:500 dilution.
- Incubate secondary antibody for 4 hr with gentle rocking at RT in the dark. From now on, process the samples under dimmed lights and incubate in a dark container or by wrapping in aluminum foil.
- At the end of the incubation, remove and discard the secondary antibody solution. Add 0.5 ml 2% NGS/PBTx to wash. Gently rock for 5 min. Remove and discard solution. Repeat twice.
- If co-immunostaining, obtain the second primary antibody from a different species than the first primary antibody. In these studies, follow the rabbit anti-phospho CaMK-II antibody by the red-fluorescent dye-conjugated goat anti-rabbit IgG secondary antibody.
- Dilute rabbit anti-phospho CaMK-II antibody 1:20. For each tube of embryos, suspend in 0.3 ml 10% NGS/PBTx, then add 15 μl of the stock antibody solution.
- Ensure that embryos are immersed and lights are dimmed. Incubate with gentle rocking O/N at RT in the dark.
- In the morning under dim lights, remove and discard antibody solution. Add 0.5 ml 2% NGS/PBTx. Gently rock for 5 min. Remove and discard solution. Repeat twice.
- Keep overhead lights dim and dilute enough of the appropriate fluorescently-conjugated secondary antibody so that each condition contains at least 0.5 ml. In this study, dilute the red-fluorescent dye-conjugated goat anti-rabbit IgG secondary antibody (red channel) 1:500 in 10% NGS/PBTx.
- Incubate second secondary antibody for 4 hr with gentle rocking at RT in the dark.
- At the end of this incubation and under dim lights, remove and discard the secondary antibody solution. Add 0.5 ml PBTx. Gently rock for 5 min. Remove and discard solution. Repeat twice. Store in either PBTx or 50% glycerol/PBS depending on the imaging procedure.
4. Confocal Imaging and Processing
- Mount embryos for imaging. Place 1 – 5 embryos on a glass slide. Create a chamber between the main coverslip and the slide using coverslip fragments on each side of the chamber. Typically, four #1 coverslips are used to make these spacer stacks without sealing.
NOTE: No mounting medium is necessary. - Use a 100X oil immersion objective to bring single embryo into focus using transmitted light. Turn off light. Turn on confocal microscope. Ensure that appropriate lasers (green and/or red) are turned on and remote focus accessary is engaged.
- Turn on the confocal program. In the "Acquire bar," select the proper objective. In the "XY Basic" bar, set image size to 1,024 by clicking the 1,024 button.
- In the "Laser and Detector" bar, click on the red 488 box and the green 568 box to turn on laser and detector for each channel. Set pinhole to medium and adjust the gain in the "Gain" bar of each channel to visualize.
- In the "Acquire Settings" bar, click "Live" to begin acquiring images. In the "View Settings" bar, uncheck the "Force Integral Zoom" box to center in the "Live" window.
- In the "Acquire Settings" bar, under the "Z" tab, step through the layers of the image. After selecting the number of optical sections to incorporate into the image, move to some point in the "center" of the z-plane.
- Under the "Z" tab, click the small red "Reference" box. This will zero the RFA at the point chosen as the "center". In the box labeled "Step Size", select the thickness of the layers (between 0.25 µm and 2.0 µm is ideal). Pay attention to the "File Size" box and try to limit to 1GB.
NOTE: Typically, up to 40 optical sections of 0.5 – 1.0 μm are obtained, but the number of sections can be determined empirically. - To find the top extreme of the image, click the circle next to "Top" and move through the z layers. Now click the circle next to "Bottom", the computer will take the image back to the layer as the center. Again move through the layers to find the bottom extreme of the image.
- Click on "Live" again in the "Acquire Settings" box to stop the laser acquisition. In this panel, click the red boxes labeled Average and Z-stack. These boxes will turn green.
- In the "Acquire Settings" box, click "Single" to acquire the entire series of images. You can monitor the progress as it scans in both channels and through the entire z-stack.
- To save, click on the volume window and click "save as" and name the file. Save as an ".ids" file. To volume render the file while the z-stack is still open, select the Data pull down menu and click on "volume render". Save the rendered file as a tiff file. This is the projected image that are shown in this publication.
Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos
Learning Objectives
Optimal Conditions for Visualizing Phospho-epitopes
Methods describing the immunolocalization of protein epitopes in zebrafish embryos have been relatively sparse compared to those for localizing mRNAs via in situ hybridization. Fixatives used in localizing protein epitopes in ciliated cells of zebrafish embryos have included 4% PFA/PBS and Dent's fixative, which is a mixture of methanol and DMSO.19 Localization of RNA by whole mount in situ hybridization (WISH) is typically achieved using PFA followed by storage in 100% methanol.20,21 Our studies revealed that the WISH fixative combination, when limiting the time in methanol to between two and seven days, was vastly superior to any other fixative for immunolocalization with the P-CaMK-II antibody. The signal achieved with this PFA/methanol combination was also significantly greater than when Dent's fixative, 4% PFA/PBS or methanol were used alone, depending on the developmental time and location within the embryo.
When optimizing the technique described here and for each fixative and developmental time used, a control sample was prepared in which all steps were followed, except that the primary antibody was omitted. Such control samples lacked a fluorescent signal when imaged under the same conditions described above. This control is recommended for other investigators replicating this approach with any antibody.
The PFA/methanol fixative combination yielded the strongest P-CaMK-II signal and was also compatible with immunostaining for acetylated tubulin, which is a standard marker for cilia. Developmentally, the first ciliated organ that emerges and has been imaged in these studies is the Kupffer's vesicle (KV). The KV is located at the posterior end of the notochord as shown at the 12-somite (15 hpf) stage (Figure 1). The KV is a transient organ and is responsible for left-right asymmetry. It emerges at the 2-somite stage (12 hpf) and disappears around the 18-somite stage. Beating primary cilia generate a circular flow of fluid, which leads to an elevation of Ca2+ in the ciliated cells lining the KV and the activation of CaMK-II in discrete locations (Figure 1). P-CaMK-II reactivity appears and disappears on one side of the KV as long as cilia are beating.12 At this early stage, total embryonic CaMK-II levels are still only half the level as at 24 hpf and one tenth of the level at 3 days of development.11 Perhaps because total CaMK-II expression is relatively low at 15 hpf, any improvement in the immunostaining technique at this stage is especially important.
Between 24 and 72 hpf of development, P-CaMK-II appears on the apical surface of ciliated cells lining specific regions of the pronephric ducts (Figures 2,3). The immunoreactivity of total CaMK-II along the entire pronephric duct and throughout adjacent somites (Figure 2) demonstrates that only a subset of embryonic CaMK-II is activated (P-CaMK-II).
Fixation Conditions are Compatible with Preserving GFP Fluorescence
These fixation techniques are also compatible with retaining green fluorescent protein (GFP) fluorescence without enhancement (Figure 3). In the GFP-tagged CaMK-II construct that is used in this figure, GFP retains sufficient fluorescence though PFA/methanol fixation and subsequent immunostaining. In a high magnification view of cells lining the pronephric duct (Figure 3), the mosaic expression of GFP-CaMK-II can be viewed in cells that have also been immunostained for P-CaMK-II.
The zebrafish embryonic ear is another tissue in which the elevation of Ca2+, acting through CaMK-II, influences its development.14 Zebrafish ears normally appear at around one day of development. At this time, CaMK-II is intensely activated at the base of the kinocilia and at lower levels along the kinocilium (Figure 4). This set of images shows that this fixation and immunolocalization technique can detect staining within cilia, a structure whose cross-section is less than 1 μm. These cilia retain their structure throughout this fixation and immunostaining process.
An additional example of two-color counterstaining in the inner ear at a later time of development was achieved by staining with both Alexa488 phalloidin and anti-acetylated tubulin followed by an Alexa568 tagged secondary antibody (Figure 5). It is noteworthy that the PFA/methanol fixation method preserves both F-actin and tubulin, which is not true of all fixatives. This example is shown as a merged color image for the entire ear and then for sub-regions.14
A final representative example of two-color counterstaining was achieved with a strain of fish that produced embryos with membrane-targeted GFP (Figure 6). Membrane targeted GFP is not attached to any other protein and is lost when extracted with methanol, therefore this image was obtained from fish that were fixed with PFA alone. This result suggests that methanol treatment extracts membranes, so it is not recommended for proteins that may be exclusively membrane bound. The P-CaMK-II signal in this figure is diminished relative to that achieved if methanol were also used, but this demonstrates that at this stage and location (kidney) of the developing embryo, the signal is strong enough to be detected without methanol treatment. That is not true at the KV stage; i.e., methanol is required to visualize P-CaMK-II immunostaining. This example is only shown as a merged image where the kidney's pronephric duct is adjacent to trunk muscle.
In summary, the PFA/methanol fixation method described here is compatible with the preservation of GFP and with staining for F-actin, acetylated tubulin, total CaMK-II and P-CaMK-II.
Figure 1. P-CaMK-II Counterstained for Acetylated Tubulin (cilia) in Zebrafish KV. Views of a single live 12 somite stage (12 ss) embryo reveals the posterior location of the KV by the arrowhead (lateral view, A) and the circle within the box (ventral view, B). Embryos at this stage were fixed using PFA/methanol. Embryos were immunostained for acetylated tubulin followed by an Alexa488-labelled secondary antibody (green channel). Next, the rabbit polyclonal antibody against P-CaMK-II was followed by an Alexa568-labelled secondary antibody (red channel). Two-channel fluorescent image projections were acquired as described at progressively higher magnifications (C,D). Scale bar = 10 μm. This figure is modified from a previous publication.12 Please click here to view a larger version of this figure.
Figure 2. P-CaMK-II Counterstained for Total CaMK-II along Zebrafish Kidney. A single dechorionated embryo at 30 hpf was fixed using PFA/methanol. Embryos were then stained for total CaMK-II followed by the Alexa488 labeled secondary antibody (green). Next, the P-CaMK-II primary antibody was followed by the Alexa568 labeled secondary antibody (red). Staining reveals an enrichment of activated CaMK-II (in red) along the apical surface of cells that line the ducts of the pronephric zebrafish kidney whereas total CaMK-II is enriched at the somite boundaries of adjacent muscle tissue and throughout sarcomeres. Scale bar = 50 μm. These images were acquired at a lower magnification than is described in the text in order to visualize the entire kidney. This figure is modified from a previous publication.13 Please click here to view a larger version of this figure.
Figure 3. P-CaMK-II Counter Imaged for GFP in Zebrafish Kidney Cells. This 30 hpf embryo was injected with a cDNA encoding a dominant negative GFP-CaMK-II.13 Such injections typically show mosaic expression. The embryo was fixed using PFA/methanol and then immunostained for P-CaMK-II using an Alexa568 labeled secondary antibody. Imaging reveals that GFP persists through PFA/methanol fixation, rehydration, blocking and immunostaining. Scale bar = 10 μm. This figure is modified from a previous publication.13 Please click here to view a larger version of this figure.
Figure 4. P-CaMK-II Counterstained with Acetylated Tubulin in Zebrafish Inner Ear. This 30 hpf embryo was fixed using PFA/methanol. The anti-acetylated tubulin antibody was followed in order by the Alexa488-labeled secondary antibody, the P-CaMK-II antibody and the Alexa568 labeled secondary antibody. Staining reveals that P-CaMK-II is present along inner ear cilia and co-localizes with acetylated tubulin. Scale bar = 5 μm. This figure is modified from a previous publication.14 Please click here to view a larger version of this figure.
Figure 5. Actin and Acetylated Tubulin in Zebrafish Inner Ear. A three day old (72 hpf) zebrafish embryo was fixed and immunostained for acetylated tubulin using Alexa568-labeled secondary antibodies, followed by actin visualization using Alexa488 phalloidin. Cilia as well as axonal innervation of the ear is revealed by acetylated tubulin (in red) and F-actin structures include muscle fibers (in green). Two ciliated sensory regions of the zebrafish ear are shown in more detail: the anterior macula (am) and posterior crista (pc). Scale bar = 10 μm. This figure is modified from a previous publication.14 Please click here to view a larger version of this figure.
Figure 6. P-CaMK-II Counter Imaged for Membrane GFP in Zebrafish Kidney. This single merged image in which the β-actin:CAAX-GFP embryo (30 hpf) was fixed and stained for P-CaMK-II followed by the Alexa568 labeled secondary antibody. Staining reveals an enrichment of activated CaMK-II (in red) along the apical surface of cells that line the ducts of the pronephric zebrafish kidney. These kidney ductal cells are nestled just underneath muscle tissue and both are marked by the expression of membrane targeted GFP (in green). Scale bar = 50 μm. Please click here to view a larger version of this figure.
List of 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" |
Lab Prep
The rapid proliferation of cells, the tissue-specific expression of genes and the emergence of signaling networks characterize early embryonic development of all vertebrates. The kinetics and location of signals – even within single cells – in the developing embryo complements the identification of important developmental genes. Immunostaining techniques are described that have been shown to define the kinetics of intracellular and whole animal signals in structures as small as primary cilia. The techniques for fixing, imaging and processing images using a laser-scanning confocal compound microscope can be completed in as few as 36 hr.
Zebrafish (Danio rerio) is a desirable organism for investigators who seek to conduct studies in a vertebrate species that is affordable and relevant to human disease. Genetic knockouts or knockdowns must be confirmed by the loss of the actual protein product. Such confirmation of protein loss can be achieved using the techniques described here. Clues into signaling pathways can also be deciphered by using antibodies that are reactive with proteins that have been post-translationally modified by phosphorylation. Preserving and optimizing the phosphorylated state of an epitope is therefore critical to this determination and is accomplished by this protocol.
This study describes techniques to fix embryos during the first 72 hr of development and co-localize a variety of relevant epitopes with cilia in the Kupffer's Vesicle (KV), the kidney and the inner ear. These techniques are straightforward, do not require dissection and can be completed in a relatively short period of time. Projecting confocal image stacks into a single image is a useful means of presenting these data.
The rapid proliferation of cells, the tissue-specific expression of genes and the emergence of signaling networks characterize early embryonic development of all vertebrates. The kinetics and location of signals – even within single cells – in the developing embryo complements the identification of important developmental genes. Immunostaining techniques are described that have been shown to define the kinetics of intracellular and whole animal signals in structures as small as primary cilia. The techniques for fixing, imaging and processing images using a laser-scanning confocal compound microscope can be completed in as few as 36 hr.
Zebrafish (Danio rerio) is a desirable organism for investigators who seek to conduct studies in a vertebrate species that is affordable and relevant to human disease. Genetic knockouts or knockdowns must be confirmed by the loss of the actual protein product. Such confirmation of protein loss can be achieved using the techniques described here. Clues into signaling pathways can also be deciphered by using antibodies that are reactive with proteins that have been post-translationally modified by phosphorylation. Preserving and optimizing the phosphorylated state of an epitope is therefore critical to this determination and is accomplished by this protocol.
This study describes techniques to fix embryos during the first 72 hr of development and co-localize a variety of relevant epitopes with cilia in the Kupffer's Vesicle (KV), the kidney and the inner ear. These techniques are straightforward, do not require dissection and can be completed in a relatively short period of time. Projecting confocal image stacks into a single image is a useful means of presenting these data.
Procédure
The rapid proliferation of cells, the tissue-specific expression of genes and the emergence of signaling networks characterize early embryonic development of all vertebrates. The kinetics and location of signals – even within single cells – in the developing embryo complements the identification of important developmental genes. Immunostaining techniques are described that have been shown to define the kinetics of intracellular and whole animal signals in structures as small as primary cilia. The techniques for fixing, imaging and processing images using a laser-scanning confocal compound microscope can be completed in as few as 36 hr.
Zebrafish (Danio rerio) is a desirable organism for investigators who seek to conduct studies in a vertebrate species that is affordable and relevant to human disease. Genetic knockouts or knockdowns must be confirmed by the loss of the actual protein product. Such confirmation of protein loss can be achieved using the techniques described here. Clues into signaling pathways can also be deciphered by using antibodies that are reactive with proteins that have been post-translationally modified by phosphorylation. Preserving and optimizing the phosphorylated state of an epitope is therefore critical to this determination and is accomplished by this protocol.
This study describes techniques to fix embryos during the first 72 hr of development and co-localize a variety of relevant epitopes with cilia in the Kupffer's Vesicle (KV), the kidney and the inner ear. These techniques are straightforward, do not require dissection and can be completed in a relatively short period of time. Projecting confocal image stacks into a single image is a useful means of presenting these data.