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

Summary

Techniques are described to immunostain phospho-epitopes in whole zebrafish embryos and then conduct two-color fluorescent confocal localization in cellular structures as small as primary cilia. The techniques for fixing and imaging can define the location and kinetics of the appearance or activation of specific proteins.

Abstract

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.

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 genes^10,11 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

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

1. 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.
2. Use 100% methanol without any supplements. Use 95% ethanol without any supplements.
3. 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.
4. 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.
5. 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.
6. Prepare 50% glycerol/PBS. Mix thoroughly equal parts of 2x PBS and 100% glycerol. Store at RT.

2. Embryo Fixation

1. 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
2. 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
3. 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.
4. 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.
5. 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

1. Place a minimum of 10 embryos for each experimental condition in capped 1.5 ml microcentrifuge tube, and label them.
2. 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.
3. 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).
4. 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.
5. 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.
6. 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.
7. 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.
8. 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.
9. 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.
10. 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.
11. 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.
12. 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.
13. Ensure that embryos are immersed and lights are dimmed. Incubate with gentle rocking O/N at RT in the dark.
14. 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.
15. 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.
16. Incubate second secondary antibody for 4 hr with gentle rocking at RT in the dark.
17. 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

1. 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.
2. 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.
3. 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.
4. 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.
5. 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.
6. 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.
7. 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.
8. 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.
9. 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.
10. 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.
11. 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.

Results

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.^1...

Discussion

The PFA/methanol method was developed in this laboratory with the primary objective of optimizing the immunolocalization of the phospho-T²⁸⁷-CaMK-II epitope during zebrafish development. This method successfully localized P-CaMK-II during the formation of several ciliated organs including the zebrafish KV,¹² inner ear¹⁴ and kidney.¹³ Particularly at the KV stage, this technique was necessary. The success of this method is likely due to a combination of a) minimization of autofl...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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 α-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 1 ml 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 RT
Tween-20	Life Technologies	85113	10% solution in water, store at RT
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"