Name: analysis of apoptosis in zebrafish embryos by whole-mount immunofluorescence to detect activated caspase 3
Uploaded: 2013-12-20T14:15:00.000+00:00
Duration: 8 min 20 s
Description: Watch this Scientific Journal Video about Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3 at JoVE.com

Funding was provided by the Huntsman Cancer Foundation (CJ, CT), American Cancer Society grant #RSG-13-025-01-CSM (RS) and NIH T32 GM007464-36 (SS).

1. Preparation of Solutions
<ol>
	<li>Prepare 500 ml&#160;of 1x&#160;PBST (1x PBS, 0.1% Tween-20), and filter sterilize. 1x PBST can be kept at room temperature (RT) indefinitely as long as there are no signs of bacterial growth.</li>
	<li>CAUTION: Prepare 50 ml of 4% paraformaldehyde (PFA) in a chemical hood by dissolving powdered PFA in 1x PBS at 60 &#176;C for 1 hr and covering in aluminum foil to protect from light. Store at 4 &#176;C for 1 month, or at -20 &#176;C for 2 years.</li>
	<li>Prepare 500 ml of fresh 1x PDT (1x PBST, 0.3% Triton-X, 1% DMSO). 1x PDT can be stored at 4 &#176;C for up to one month.</li>
	<li>Prepare 50 ml of blocking buffer (1x PBST, 10% heat-inactivated fetal bovine serum, 2% bovine serum albumin). Create 10 ml aliquots, and store at -20 &#176;C for up to 1 year. Once thawed, blocking buffer can be stored at 4 &#176;C for up to 48 hr.</li>
	<li>Prepare 100 ml of 4% methylcellulose by heating 30 ml of water to 80 &#176;C. Add 4 g of methylcellulose to the hot water, and mix using a stir bar on a room-temperature stir plate.&#160; Thorough mixing usually takes between 4 hr to overnight. Add the remainder of (unheated) water, and mix at 4 &#176;C for 1 hr. Store at RT.</li>
	<li>If using embryos between tailbud stage and 32 hpf, prepare 50 ml of 10 mg/ml pronase, and store for up to 1 week at 4 &#176;C. Otherwise, store in 10 ml aliquots at -20 &#176;C for up to 1 year.</li>
	<li>Place 50 ml of 100% methanol at -20 &#176;C.</li>
	<li>Prepare 60x&#160;egg water by dissolving 280 g Instant Ocean Sea Salt in 2 L of distilled water (as in Westerfield16). Prepare 1x egg water by diluting the 60x&#160;stock in deionized water.</li>
</ol>
2. Preparation of Embryos
<ol>
	<li>Collect zebrafish embryos in a Petri dish in 1x&#160;egg water between the 4-cell stage and 32 hpf.
	<ol>
		<li>For embryos between the 4-cell and tailbud stages, proceed to step 2.2.</li>
		<li>For embryos between tailbud stage and 32 hpf, remove chorions by adding 100 &#956;l&#160;of 10 mg/ml pronase to embryos in 10 ml of 1x&#160;egg water.</li>
		<li>Note: Pronase activity can be variable between batches. Thus, final pronase concentration should be optimized for dechorionation when new batches are obtained.
		<ol>
			<li>Let incubate at room temperature for 30 min.</li>
			<li>Pour off egg water / pronase solution into waste, and slowly add back fresh 1x egg water.</li>
			<li>Rinse 2x by pouring off existing egg water (and free-floating chorions), and gently add new egg water.</li>
			<li>Note: Most chorions should be removed at this point. With a transfer pipet, gently suck embryos up and down until remaining chorions are removed.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Transfer up to 40 embryos to a 1.5 ml tube. Rinse 2 times with 1 ml of 1x PBST. &#160;</li>
	<li>Remove PBST, leaving liquid just above embryos (approximately 100 &#956;l&#160;volume should remain in the tube). Add 1 ml of 4% PFA. Lay tubes on their side (so embryos spread out along length of tube) in an appropriate-size container for ease of transport (e.g. a Petri dish or a pipet box top), and place on a gentle rocker at 4 &#176;C overnight or up to a week. &#160;
	<ol>
		<li>For embryos between the 4-cell and tailbud stages, manually dechorionate the fixed embryos with forceps.</li>
	</ol>
	</li>
</ol>
3. Immunofluorescence Procedure
<ol>
	<li>Place tubes in a tube rack so that all embryos sink to the bottom of the tube. Remove PFA (to appropriate waste container) leaving liquid just above level of embryos. Following fixation (step 2.3), embryos should never be exposed to air in order to prevent background antibody staining.</li>
	<li>Add 1 ml of ice-cold 100% methanol (to permeabilize) drop-wise. Embryos are now fragile after the fixation, and extra care must be taken to prevent them from tumbling within the tube during all subsequent removal/addition of liquids. Lay tubes on their sides, and place at -20 &#176;C for 2 hr. Alternatively, embryos can be left at -20 &#176;C for up to 24 hr.</li>
	<li>Place tubes in a rack to collect embryos at the bottom of the tube. Remove methanol, leaving liquid just above level of embryos.</li>
	<li>Carefully add 1 ml 1x PDT. Remove PDT, and add 1 ml of fresh 1x PDT. Lay tubes on their sides and gently rock at RT for 30 mins. Remove PDT, and repeat 30&#160;min wash with fresh PDT.</li>
	<li>Remove PDT until liquid is just above the level of the embryos, and add 500 &#956;l&#160;blocking buffer.
	<ol>
		<li>Lay embryos on their side in a transport container, and ensure that embryos do not travel more than 2/3 up the length of the tube. Otherwise, embryos get caught in the lid of the tube and outside of the liquid. &#160;</li>
		<li>Carefully place transport container with tubes on a very-gentle rocker at RT for 1 hr.</li>
	</ol>
	</li>
	<li>Stand tubes upright in a tube rack, and open all tubes to check for embryos stuck on the lid. For any tubes with embryos on the lid, close the top, and gently rock the tube by hand to bring all embryos back into the liquid.</li>
	<li>Add 1 &#956;l&#160;rabbit anti-activated Caspase 3 antibody (1:500 dilution).
	<ol>
		<li>If performing a colabeling experiment, simply add the appropriate dilution of the second nonrabbit-derived antibody in addition to the anti-activated Caspase 3 antibody.</li>
	</ol>
	</li>
	<li>Carefully lay embryos on their side, and gently rock at room temperature for 2 hr. Alternatively, embryos can be incubated at 4 &#176;C overnight.</li>
	<li>Stand tubes in a tube rack. Remove block/antibody solution. Add 1 ml of 1x PDT. Be sure to invert tube by hand to collect any embryos stuck on the lid of the tube. Then, open all tubes to ensure all embryos have been removed from the lid.</li>
	<li>Repeat step 3.4.</li>
	<li>Repeat steps 3.5-3.10, but substitute the anti-activated Caspase 3 antibody with the appropriate dilution of an anti-rabbit fluorophore-conjugated antibody that absorbs blue light and emits green light. However, any fluorophore-conjugated antibody with a user-preferable absorption/emission spectra can be used here. After addition of the secondary antibody, it is important to keep embryos in the dark for the remainder of the assay. Cover the transport container with aluminum foil during the incubation steps, but do not worry about light exposure when changing buffers.
	<ol>
		<li>If performing a colabeling experiment, simply add the appropriate dilution of a complementary fluorophore-conjugated secondary antibody at this step.</li>
		<li>Note: If the user wishes to colabel active-Caspase 3 with Gfp-expressing cells, then the rabbit anti-active Caspase 3 antibody should be followed here by a nongreen-fluorescing anti-rabbit secondary antibody, and the anti-GFP antibody should be followed by a green-fluorescing nonrabbit secondary antibody. This will eliminate confusion if some Gfp fluorescence remains visible after fixation of embryos.</li>
	</ol>
	</li>
</ol>
4. Analysis of Apoptotic Cells by Fluorescence Microscopy
<ol>
	<li>Analyze embryos in 1x PDT in a small Petri dish or glass spot plate under a fluorescent microscope using the appropriate light filter. If positive controls have bright fluorescence with minimal background, then proceed to step 4.2. (If not, consult the troubleshooting section in the Discussion, and repeat the protocol with new embryos.) Embryos can be stored at 4 &#176;C in 1x PDT for up to 48 hr.</li>
	<li>Pour 4% methylcellulose in a 3.5 cm Petri dish, just enough to cover the bottom of the dish. Alternatively, a glass spot plate or depression slide can be used with a large drop of methylcellulose in the depression. Carefully suck embryos from the bottom of the tube into a clean transfer pipet and transfer to the Petri plate for visualization.</li>
	<li>Orient an embryo of interest for documentation using an angled probe. There will likely be some variability between embryos from the same sample, so it is important to take images that represent fluorescence intensity and pattern observed in the majority of the embryos from that sample. Documentation should be performed within 48 hr of completing the assay to prevent accumulation of non-specific background fluorescence. Residual 4% methylcellulose will not interfere with subsequent PCR reactions required for genotyping of embryos.</li>
	<li>Note: To aid in immobilization of an embryo at a certain angle, it is helpful to gently push the embryo to the bottom of the dish so that the yolk or some other part of the embryo (depending on the desired view) is resting on the bottom of the dish. Then, use the probe to move either the embryo or the immediately surrounding methylcellulose (while simultaneously visualizing under the microscope) to obtain the desired angle for documentation.</li>
</ol>

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The authors have nothing to disclose.

The Casp3 assay as presented in this protocol is meant to be straightforward, and if performed as instructed herein, should give rise to a robust signal-to-noise ratio. The critical steps of this protocol include the use of fresh 1x&#160;PDT buffer for all washes, sufficient length of wash steps in PDT buffer following incubation of embryos in the primary antibody, gentle handling of embryos, and meticulous accounting for all embryos within tubes or transfer pipets during all steps of the protocol.
In our experience, the following are the most common reasons for lack of a signal on positive controls: 1) 1x PBST is substituted for 1x PDT during washing steps, 2) 1x PDT buffer is older than one&#160;month, 3) the wrong secondary antibody was used, 4) embryos are older than 32 hpf and/or 5) more than 40 embryos were analyzed in one tube. When inconsistent results or variability arises within samples, this is usually because 1) care was not taken to ensure that all embryos remained within liquid at all times during the procedure, and/or 2) embryos were not incubated under sufficient rocking. High levels of background noise are often visualized as a general fluorescent haze arising throughout the animal tissue of the embryo (see Figure 4, &#8220;background fluorescence&#8221;). A poor signal-to-noise ratio can arise when 1) embryos were not washed for sufficient time following the primary antibody, and/or 2) embryos were left in PFA or methanol for longer than the recommended time. If embryos fail to remain intact by the end of the assay: 1) solutions were not added drop-wise following fixation with PFA, and/or 2) rocker speed during incubation steps was set too high.
The whole-mount visualization inherent in the Casp3 assay is helpful for spatial localization of apoptotic cells. However, tissue-specificity is approximate and will likely rely on colabeling techniques (see Figure 5) and/or embryo sectioning to pinpoint the exact identity of cells undergoing apoptosis. In the protocol described here, colabeling is limited by the requirement for the specific rabbit-derived antibody used in our assay. We have not identified a commercially available nonrabbit-derived anti-activated Caspase 3 antibody that specifically labels apoptotic cells by whole-mount immunofluorescence in zebrafish embryos. Thus, cell-type specific antibodies that are raised in rabbits will not be compatible with the Casp3 assay for colabeling experiments. One alternative is to perform a TUNEL assay which requires a mouse-derived antibody to label apoptotic cells6. However, we have found that the whole-mount TUNEL assay performed on zebrafish embryos is very sensitive to a number of variables in the reaction, including the concentration of TdT enzyme, duration of embryo incubation in TdT enzyme, and the number of embryos present per tube (our unpublished observations). Thus, it is challenging to find a balance of enzyme that gives consistent interpretation of results. Therefore, a preferable alternative is to analyze apoptotic cells in transgenic zebrafish embryos that express a fluorophore (with commercially available robust antibodies) like GFP in a specific cell type. Then, colabeling experiments can be performed using a primary mouse anti-Gfp antibody followed by a green-fluorescing anti-mouse secondary antibody. See Figure 5 for an example.
While the Casp3 protocol gives robust, reliable staining of apoptotic cells in embryos from the 4-cell stage through 32 hpf, it has limited application beyond 32 hpf due to the inability of the antibodies to penetrate the skin of older embryos. Additional techniques to permeabilize the embryos, including treatment with collagenase, trypsin, acetone, or ethanol, could potentially be optimized to adapt this assay to the analysis of older embryos. Staining with AO, however, can be performed on live zebrafish up to at least 5 days-old9. Thus, the AO assay circumvents the embryonic stage restriction and represents a rapid assay to analyze apoptotic cells (1-2 hr). By contrast, the Casp3 assay requires overnight fixation of embryos and takes 2-4 days to complete. The disadvantages of the AO assay include that efficient photographic documentation of embryos can be challenging with multiple samples since embryos are alive and apoptotic cells are rapidly formed and cleared. This can be especially problematic if embryos need to be mounted to obtain a specific orientation for documentation. Additionally, we have found that AO staining diminishes after fixation with PFA, so it is not compatible with colabeling experiments involving immunofluorescence.
Quantitative analysis of activated-Caspase 3 can be performed utilizing software programs that measure fluorescence intensity of documented images. We have quantified IR-induced apoptosis in the neural tissue, a method we have described in detail elsewhere10. Depending on the experiment, quantitative analysis of activated-Caspase 3 may require further manipulation of embryos, such as flat-mounting, to obtain proper orientation for optimal tissue analysis. In addition, the linearity of the analysis will depend on the thickness of the tissue being analyzed. For instance, we analyzed IR-induced apoptosis in neural tissue by measuring active-Caspase 3 immunofluorescence in the shallow neural tissue of the spinal cord rather than the neuron-dense tissue of the brain4,10.
In our experience, the Casp3 assay is the most robust and reliable assay available to analyze apoptosis in zebrafish embryos between the 4-cell stage and 32 hpf when analysis requires ample time for documentation. When appropriate antibodies or transgenic lines are available to identify specific cells, this assay is also highly amenable to colabeling to identify the specific cell types undergoing apoptosis.

Here we describe how to perform whole-mount immunofluorescence in early zebrafish embryos to detect cells with activated Caspase 3 (referred to hereafter as the Casp3 assay). The overall goal of this method is to identify the relative abundance and location of apoptotic cells in zebrafish from the 4-cell stage to 32 hr-post-fertilization (hpf).
Both intrinsic (mitochondria-mediated) and extrinsic (death-receptor-mediated) apoptotic pathways mediate cell death through activation of the caspase cascade1-2. Initiator caspases normally exist as monomers within the cell, and once recruited to pathway-specific platforms in response to a pro-apoptotic signal, they become activated dimers. Activated initiator caspases (Caspase 9; intrinsic pathway, and Caspase 8; extrinsic pathway) then target the predimerized, inactive effector caspases (Caspase 3, 6, 7) for cleavage to form active heterotetramers. Active effector caspases then cleave cellular targets with specific tetrapeptide residues to initiate the specific proteolytic destruction that culminates in the ultrastructural hallmarks of apoptosis, such as chromatin condensation and membrane blebbing, and cell surface signals that mediate packaging of the cell for engulfment by phagocytes in an immune silent manner. In mammalian cells, active effector caspases are thought to perform the majority of cellular cleavage events that mediate apoptosis through both the intrinsic and extrinsic pathways1-2. The protocol described herein uses an antibody that specifically binds the cleaved, heterotetrameric form of Caspase 3, but not the inactive Caspase 3 dimer.
One of the advantages of the Casp3 assay is the ability to visualize the spatial distribution of apoptotic cells within the context of the whole embryo. Therefore, tissues of interest can be examined for the presence of apoptotic cells, and if antibodies or fluorescent transgenic strains exist that label specific cell types, then it is possible to pinpoint the exact identity of cells undergoing apoptosis through colabeling experiments. Several groups have successfully used activated Caspase 3 as a marker for apoptosis in zebrafish embryos (e.g. 3-7). Additional whole-mount techniques to identify apoptotic cells in zebrafish embryos include the TUNEL assay8 and acridine orange (AO) staining9. We have used all three methods to identify apoptotic cells in zebrafish embryos4,6,10.
TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) staining takes advantage of the Terminal deoxynucleotidyl Transferase (TdT) enzyme that is used to add nucleotides to 3&#8217; hydroxyl ends of DNA during VDJ recombination in lymphocytes11. TdT adds dUTP-biotin to free 3&#8217; hydroxyl groups in the DNA, which are abundant primarily in apoptotic cells. Tissues are then exposed to avidin-peroxidase, and apoptotic cells are stained upon addition of the substrate 3-amino-9-ethylcarbazole. The TUNEL assay was used to perform an extensive analysis of apoptotic cells during embryonic development in wild-type zebrafish tissues12. Similar to the Casp3 assay, the TUNEL assay is only performed on fixed embryos. We have found the Casp3 assay to be more consistent and less expensive than the TUNEL assay.
AO staining was first described as a mechanism to specifically identify apoptotic cells in Drosophila13. AO stains both the cytoplasm and nucleus of apoptotic, but not necrotic, cells. AO is well known to bind nucleic acids14, and this is thought to account for the nuclear staining. However, AO does not stain the chromatin of live cells; rather, it appears that specific changes occurring during apoptosis permit intercalation and staining of condensed apoptotic nuclei15. AO staining has the advantage of rapid apoptotic analysis of live embryos. However, documentation of samples must be performed quickly due to the dynamic nature of apoptotic cell formation and clearance. On the other hand, simultaneous fixation of all samples provides a snapshot of apoptosis at a given time across all samples and is not subject to this temporal variable. Thus, since analysis of apoptotic cells by AO staining is not compatible with fixed embryos, the Casp3 assay is preferable for experiments in which photographic documentation and/or quantitation is required across multiple samples.

<table><tbody><tr><td>1.5 ml tubes&amp;nbsp;</td><td>Denville Scientific</td><td>c2170</td><td>Nonhazardous. Any similar 1.5 m plastic tubes will suffice.</td></tr><tr><td>10x Phosphate Buffered Saline (PBS)</td><td>Sigma Aldrich</td><td>P5493</td><td>Nonhazardous.</td></tr><tr><td>a-activated-human-Caspase-3 antibody</td><td>Fisher Scientific</td><td>BDB559565</td><td>May be harmful if inhaled or swallowed.</td></tr><tr><td>a-GFP monoclonal antibody (mouse)</td><td>Life Technologies</td><td>33-2600</td><td>May be harmful if inhaled or swallowed.</td></tr><tr><td>a-HuC/HuD human neuronal protein, mouse IgG2b, monoclonal 16A11 - UNCONJ</td><td>Life Technologies</td><td>A21271</td><td>May be harmful if inhaled or swallowed.</td></tr><tr><td>Albumin, Bovine BSA Heat Shock Isolation&amp;nbsp;</td><td>Amresco</td><td>CAS 9048-46-8</td><td>Nonhazardous. Can also be purchased through Bioexpress: 0332-1006</td></tr><tr><td>Alexa Fluor 488 Donkey anti-mouse IgG</td><td>Life Technologies</td><td>A11029</td><td>Use at 1:200 dilution.&amp;nbsp; May be harmful if inhaled or swallowed.</td></tr><tr><td>Alexa Fluor 488 Donkey anti-Rabbit IgG</td><td>Life Technologies</td><td>A21206</td><td>Use at 1:200 dilution.&amp;nbsp; May be harmful if inhaled or swallowed.</td></tr><tr><td>Alexa Fluor 568 Donkey anti-Rabbit IgG</td><td>Life Technologies</td><td>A10042</td><td>Use at 1:200 dilution.&amp;nbsp; May be harmful if inhaled or swallowed.</td></tr><tr><td>Dimethyl Sulfoxide (DMSO)</td><td>Sigma Aldrich</td><td>D8418</td><td>Flammable.</td></tr><tr><td>Dumont #5 Inox Forceps (standard tips/straight)</td><td>Fine Science Tools</td><td>11251-20</td><td>Sharp!</td></tr><tr><td>FBS HyClone Fetal Bovine Serum&amp;nbsp;</td><td>Thermo-Fisher</td><td>SH3091003</td><td>Nonhazardous.</td></tr><tr><td>Fisher Scientific Ocelot Rotator</td><td>Fisher Scientific</td><td>05-450-21</td><td>Any rotator with manual speed control is suitable.</td></tr><tr><td>Instant Ocean Aquarium Salt&amp;nbsp;</td><td>Petco</td><td>1373684</td><td>Nonhazardous.</td></tr><tr><td>Methanol (acetone-free)</td><td>Sigma Aldrich</td><td>M1775</td><td>Flammable: toxic by inhalation, ingestion and contact.</td></tr><tr><td>Methylcellulose</td><td>Sigma Aldrich</td><td>M0387-1006</td><td>Nonhazardous.</td></tr><tr><td>Paraformaldehyde</td><td>Sigma Aldrich</td><td>P6148</td><td>Flammable: toxic by inhalation, ingestion and contact.</td></tr><tr><td>Probe (Angled 80 Short/15.5cm/0.15mm tip diameter)</td><td>Fine Science Tools</td><td>10140-02</td><td>Sharp!</td></tr><tr><td>Pronase</td><td>Roche</td><td>11459643001</td><td>Nonhazardous.</td></tr><tr><td>Pyrex Spot Plate</td><td>Fisher Scientific</td><td>13-748B</td><td>Nonhazardous.</td></tr><tr><td>Transfer Pipets, Samco general purpose</td><td>Thermo-Scientific</td><td>204</td><td>Nonhazardous.</td></tr><tr><td>Triton X-100</td><td>Sigma Aldrich</td><td>T8787</td><td>Irritant: harmful by ingestion or contact.</td></tr><tr><td>Tween-20</td><td>Sigma Aldrich</td><td>P7949</td><td>Nonhazardous. Other brands include VWR #97063-872.</td></tr></tbody></table>

analysis of apoptosis in zebrafish embryos by whole-mount immunofluorescence to detect activated caspase 3

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. The whole-mount analysis provides spatial information in regard to tissue specificity of apoptosing cells, although sectioning and/or colabeling is ultimately required to pinpoint the exact cell types undergoing apoptosis. The whole-mount Casp3 assay is optimized for analysis of fixed embryos between the 4-cell stage and 32 hr-post-fertilization and is useful for a number of applications, including analysis of zebrafish mutants and morphants, overexpression of mutant and wild-type mRNAs, and exposure to chemicals. Compared to acridine orange staining, which can identify apoptotic cells in live embryos in a matter of hours, Casp3 and TUNEL assays take considerably longer to complete (2-4 days). However, because of the dynamic nature of apoptotic cell formation and clearance, analysis of fixed embryos ensures accurate comparison of apoptotic cells across multiple samples at specific time points. We have also found the Casp3 assay to be superior to analysis of apoptotic cells by the whole-mount TUNEL assay in regard to cost and reliability. Overall, the Casp3 assay represents a robust, highly reproducible assay in which to analyze apoptotic cells in early zebrafish embryos.

The Casp3 assay gives clear evidence of apoptotic cells from the 4-cell stage to 32 hpf. Figure 2A shows that injection of embryos with high concentrations of mRNA encoding potent pro-apoptotic bh3-only proteins, such as Bim or Noxa, quickly gives rise to active-Caspase 3 by the 4-cell stage of development. Embryonic death subsequently occurs within 1 hr4. Note that intensity of fluorescence is moderate, but results are conclusive. At older stages, fluorescence intensity is more robust. For instance, Figure 2B shows bright active-Caspase 3-positive cells in sphere-stage embryos that have been injected with moderately toxic levels4 of mRNA encoding the pro-apoptotic bh3-only gene puma. Messenger RNA encoding the anti-apoptotic bcl-2 gene was coinjected to prove that injection of puma mRNA induces apoptosis through the intrinsic, mitochondria-mediated pathway.
IR-induced apoptosis in zebrafish requires p53-mediated transcription of bh3-only genes such as puma5-6. The delay in IR-induced apoptosis in Figure 3 is consistent with the requirement for p53-mediated transcription.
The Casp3 protocol should give rise to a robust signal-to-noise ratio. Figure 4 compares the optimal Casp3 assay result to two common suboptimal results that arise when critical steps are compromised (see Discussion): A weak signal-to-noise ratio due to background fluorescence in animal tissue and yolk fluorescence that distracts from the signal-of-interest.
Colabeling experiments using transgenic GFP-expressing lines approximate tissues undergoing apoptosis (Figure 5).
<img alt="Immunostaining workflow for embryo analysis; diagram shows antibody application and visualization steps." fo:content-width="3in" fo:src="/files/ftp_upload/51060/51060fig1highres.jpg" src="/files/ftp_upload/51060/51060fig1.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1. Simplified schematic of the Casp3 protocol.
<img alt="Embryo developmental stages in bright-field; comparison of control vs gene expressions for apoptosis." fo:content-width="5in" fo:src="/files/ftp_upload/51060/51060fig2highres.jpg" src="/files/ftp_upload/51060/51060fig2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2. (A) Analysis of apoptosis in 4-cell stage embryos. Embryos were injected at the one-cell stage of development with 50 pg of mRNA encoding egfp (control) or the pro-apoptotic bh3-only genes bim and noxa. At the 4-cell stage (approximately one hr-post-injection, hpi), embryos were analyzed by immunofluorescence to detect activated Caspase 3. Bim and noxa mRNA induce rapid apoptosis in zebrafish embryos immediately following injection which, if embryos are left to develop, leads to death by 2 hpi4. A brightfield image of a wild-type 4-cell stage embryo is shown for comparison. (B) Analysis of apoptosis in sphere-stage zebrafish embryos. Embryos were injected at the one-cell stage of development with mRNA encoding either 50 pg egfp (control), 5 pg puma plus 45 pg egfp (puma), or 5 pg puma plus 45 pg bcl-2 (bcl-2). At sphere stage (approximately 4 hpi), embryos were analyzed by the Casp3 assay. Both lateral and animal pole views are shown to visualize apoptotic cells. Rescue of puma-induced apoptosis by bcl-2 indicates that injection of puma mRNA induces apoptosis specifically through the intrinsic, mitochondria-mediated pathway. A brightfield image of a wild-type sphere-stage embryo is shown for comparison.
<img alt="Embryo development stages, bright-field and infrared images, highlighting cellular progression." fo:content-width="5in" fo:src="/files/ftp_upload/51060/51060fig3highres.jpg" src="/files/ftp_upload/51060/51060fig3.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3. Time course of ionizing radiation (IR)-induced apoptosis between 17-20 hpf. Zebrafish embryos were irradiated with 8 Gy IR at 17 hpf and analyzed at 0, 1.5, 2, and 3 hr-post-IR (hpIR) by the Casp3 assay. Arrows at 20 hpf show accumulation of apoptotic cells in the spinal cord at 3 hpIR. A brightfield image of a 17-hpf embryo is shown for comparison.
<img alt="Zebrafish embryo fluorescence analysis; comparative imaging pre, post IR; examines optimal fluorescence." fo:content-width="4in" fo:src="/files/ftp_upload/51060/51060fig4highres.jpg" src="/files/ftp_upload/51060/51060fig4.jpg" data-altupdatedat="1747911167000" data-alt="Figure 4"> 
Figure 4. Optimal and sub-optimal Casp3 immunofluorescence results on 27-hpf embryos. Zebrafish embryos were irradiated (or not) at 24 hpf with 15 Gy and analyzed by the Casp3 assay. The "optimal" group shows ideal results in which apoptotic cells brightly fluoresce, and nonapoptotic cells and the yolk show minimal background fluorescence.  Arrows in the “background fluorescence in control” group denote excessive background fluorescence in the animal tissue of control embryos. Asterisks in the “yolk fluorescence” group demonstrate excessive nonspecific fluorescence in the yolk.
<img alt="Fluorescence microscopy overlay showing α-Gfp and α-Casp3 expression in different Tg lines." fo:content-width="5in" fo:src="/files/ftp_upload/51060/51060fig5highres.jpg" src="/files/ftp_upload/51060/51060fig5.jpg" data-altupdatedat="1747911167000" data-alt="Figure 5"> 
Figure 5. Colabeling experiments approximate tissue specificity of IR-induced apoptosis. The Tg(olig2:egfp)vu12 transgenic line labels motor neurons and oligodendrocytes17, and the Tg(elavl3:egfp) line labels all differentiating neurons18. Each transgenic line was irradiated with 15 Gy at 24 hpf and analyzed 3 hr later using both mouse anti-GFP and rabbit anti-active-Caspase 3 primary antibodies, followed by green- and red-fluorescing secondary antibodies, respectively. A magnification of the spinal cord (located dorsal to the end of the yolk extension of 27-hpf, 3-hpIR embryos) is shown. Apoptotic cells appear to be partially localized to both Tg(elavl3:egfp) and Tg(olig2:egfp)vu12 populations (see arrowheads in overlay), suggesting that neurons and oligodendrocytes are sensitive to IR-induced apoptosis.

Watch this Scientific Journal Video about Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3 at JoVE.com

Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3

Certain genetic perturbations or exposure to toxins can disrupt normal developmental processes leading to death of specific cell types. The analysis of activated Caspase 3 by whole-mount immunofluorescence in zebrafish embryos reveals stage- and tissue-specific localization of cells specifically undergoing apoptosis.

Analisi di apoptosi in embrioni di zebrafish da Whole-mount immunofluorescenza per rilevare caspasi 3 attivata

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis ...

analysis-apoptosis-zebrafish-embryos-whole-mount-immunofluorescence

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JoVE Journal

Biology

19.8K Views.  University of Utah. Certain genetic perturbations or exposure to toxins can disrupt normal developmental processes leading to death of specific cell types. The analysis of activated Caspase 3 by whole-mount immunofluorescence in zebrafish embryos reveals stage- and tissue-specific localization of cells specifically undergoing apoptosis.

Video: Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3

Observing Mitotic Division and Dynamics in a Live Zebrafish Embryo

Mitosis is critical for organismal growth and differentiation. The process is highly dynamic and requires ordered events to accomplish proper chromatin condensation, microtubule-kinetochore attachment, chromosome segregation, and cytokinesis in a small time frame. Errors in the delicate process can result in human disease, including birth defects and cancer. Traditional approaches investigating human mitotic disease states often rely on cell culture systems, which lack the natural physiology and developmental/tissue-specific context advantageous when studying human disease. This protocol overcomes many obstacles by providing a way to visualize, with high resolution, chromosome dynamics in a vertebrate system, the zebrafish. This protocol will detail an approach that can be used to obtain dynamic images of dividing cells, which include: in vitro transcription, zebrafish breeding/collecting, embryo embedding, and time-lapse imaging. Optimization and modifications of this protocol are also explored. Using H2A.F/Z-EGFP (labels chromatin) and mCherry-CAAX (labels cell membrane) mRNA-injected embryos, mitosis in AB wild-type, auroraBhi1045, and esco2hi2865 mutant zebrafish is visualized. High resolution live imaging in zebrafish allows one to observe multiple mitoses to statistically quantify mitotic defects and timing of mitotic progression. In addition, observation of qualitative aspects that define improper mitotic processes (i.e., congression defects, missegregation of chromosomes, etc.) and improper chromosomal outcomes (i.e., aneuploidy, polyploidy, micronuclei, etc.) are observed. This assay can be applied to the observation of tissue differentiation/development and is amenable to the use of mutant zebrafish and pharmacological agents. Visualization of how defects in mitosis lead to cancer and developmental disorders will greatly enhance understanding of the pathogenesis of disease.

Mitosis is critical to every living organism and defects often lead to cancer and developmental disorders. Using this imaging protocol and zebrafish as a model system, researchers can visualize mitosis in a live vertebrate organism and the multitude of defects that arise when mitotic processes are defective.

Osservando divisione mitotica e Dynamics in un embrione vivo Zebrafish

Mitosis is critical for organismal growth and differentiation. The process is highly dynamic and requires ordered events to accomplish proper chromatin ...

Ricerca

Biologia dello sviluppo

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 ...

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. ...

Using Immunofluorescence to Detect PM2.5-induced DNA Damage in Zebrafish Embryo Hearts

Ambient fine particulate matter (PM2.5) exposure can lead to cardiac developmental toxicity but the underlying molecular mechanisms are still unclear. 8-hydroxy-2&#39;deoxygenase (8-OHdG) is a marker of oxidative DNA damage and &#947;H2AX is a sensitive marker for DNA double strand breaks. In this study, we aimed to detect PM2.5-induced 8-OHdG and &#947;H2AX changes in the heart of zebrafish embryos using an immunofluorescence assay. Zebrafish embryos were treated with extractable organic matters (EOM) from PM2.5 at 5 &#956;g/mL in the presence or absence of antioxidant N-acetyl-L-cysteine (NAC, 0.25 &#956;M) at 2 h post fertilization (hpf). DMSO was used as a vehicle control. At 72 hpf, hearts were dissected from embryos using a syringe needle and fixed and permeabilized. After being blocked, samples were probed with primary antibodies against 8-OHdG and &#947;H2AX. Samples were then washed and incubated with secondary antibodies. The resulting images were observed under fluorescence microscopy and quantified using ImageJ. The results show that EOM from PM2.5 significantly enhanced 8-OHdG and &#947;H2AX signals in the heart of zebrafish embryos. However, NAC, acting as a reactive oxygen species (ROS) scavenger, partially counteracted the EOM-induced DNA damage. Here, we present an immunofluorescence protocol for investigating the role of DNA damage in PM2.5-induced heart defects that can be applied to the detection of environmental chemical-induced protein expression changes in the hearts of zebrafish embryos.

Using Immunofluorescence to Detect PM2.5-induced DNA Damage in Zebrafish Embryo Hearts

This protocol uses an immunofluorescence assay to detect PM2.5-induced DNA damage in the dissected hearts of zebrafish embryos.

Utilizzo dell'immunofluorescenza per rilevare il danno al DNA indotto da PM2,5nei cuori embrionali di zebrafish

Ambient fine particulate matter (PM2.5) exposure can lead to cardiac developmental toxicity but the underlying molecular mechanisms are still unclear. ...

Live Imaging of Cell Extrusion from the Epidermis of Developing Zebrafish

Homeostatic maintenance of epithelial tissues requires the continual removal of damaged cells without disrupting barrier function. Our studies have found that dying cells send signals to their live neighbors to form and contract a ring of actin and myosin that ejects it out from the epithelial sheet while closing any gaps that might have resulted from its exit, a process termed cell extrusion1. The optical clarity of developing zebrafish provides an excellent system to visualize extrusion in living epithelia. Here we describe a method to induce and image extrusion in the larval zebrafish epidermis. To visualize extrusion, we inject a red fluorescent protein labeled probe for F-actin into one-cell stage transgenic zebrafish embryos expressing green fluorescent protein in the epidermis and induce apoptosis by addition of G418 to larvae. We then use time-lapse imaging on a spinning disc confocal microscope to observe actin dynamics and epithelial cell behaviors during the process of apoptotic cell extrusion. This approach allows us to investigate the extrusion process in live epithelia and will provide an avenue to study disease states caused by the failure to eliminate apoptotic cells.

Dying cells are extruded from epithelial tissues by concerted contraction of neighboring cells without disrupting barrier function. The optical clarity of developing zebrafish provides an excellent system to visualize extrusion in living epithelia. Here we describe methods to induce and image extrusion in the larval zebrafish epidermis at cellular resolution.

Immagini dal vivo di estrusione in cella dal Epidermide di Zebrafish in via di sviluppo

Homeostatic maintenance of epithelial tissues requires the continual removal of damaged cells without disrupting barrier function.  Our studies have found ...

Biologia

A Zebrafish Embryo Model for In Vivo Visualization and Intravital Analysis of Biomaterial-associated Staphylococcus aureus Infection

Biomaterial-associated infection (BAI) is a major cause of the failure of biomaterials/medical devices. Staphylococcus aureus is one of the major pathogens in BAI. Current experimental BAI mammalian animal models such as mouse models are costly and time-consuming, and therefore not suitable for high throughput analysis. Thus, novel animal models as complementary systems for investigating BAI in vivo are desired. In the present study, we aimed to develop a zebrafish embryo model for in vivo visualization and intravital analysis of bacterial infection in the presence of biomaterials based on fluorescence microscopy. In addition, the provoked macrophage response was studied. To this end, we used fluorescent protein-expressing S. aureus and transgenic zebrafish embryos expressing fluorescent proteins in their macrophages and developed a procedure to inject bacteria alone or together with microspheres into the muscle tissue of embryos. To monitor bacterial infection progression in live embryos over time, we devised a simple but reliable method of microscopic scoring of fluorescent bacteria. The results from microscopic scoring showed that all embryos with more than 20 colony-forming units (CFU) of bacteria yielded a positive fluorescent signal of bacteria. To study the potential effects of biomaterials on infection, we determined the CFU numbers of S. aureus with and without 10 &#181;m polystyrene microspheres (PS10) as model biomaterials in the embryos. Moreover, we used the ObjectJ project file &#34;Zebrafish-Immunotest&#34; operating in ImageJ to quantify the fluorescence intensity of S. aureus infection with and without PS10 over time. Results from both methods showed higher numbers of S. aureus in infected embryos with microspheres than in embryos without microspheres, indicating an increased infection susceptibility in the presence of the biomaterial. Thus, the present study shows the potential of the zebrafish embryo model to study BAI with the methods developed here.

A Zebrafish Embryo Model for In Vivo Visualization and Intravital Analysis of Biomaterial-associated Staphylococcus aureus Infection

The present study describes a zebrafish embryo model for in vivo visualization and intravital analysis of biomaterial-associated infection over time based on fluorescence microscopy. This model is a promising system complementing mammalian animal models such as mouse models for studying biomaterial-associated infections in vivo.

Un modello di embrione di Zebrafish per nel Vivo visualizzazione e analisi videomicroscopia del biomateriale-collegata dello stafilococco aureo infezione

Biomaterial-associated infection (BAI) is a major cause of the failure of biomaterials/medical devices. Staphylococcus aureus is one of the major ...

Bioingegneria

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, ...

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model congenital abnormalities. During the first day of life, the zebrafish embryo progresses through many developmental stages including fertilization, cleavage, gastrulation, segmentation, and the organogenesis of structures such as the kidney, heart, and central nervous system. The anatomy of a young zebrafish embryo presents several challenges for the visualization and analysis of the tissues involved in many of these events because the embryo develops in association with a round yolk mass. Thus, for accurate analysis and imaging of experimental phenotypes in fixed embryonic specimens between the tailbud and 20 somite stage (10 and 19 hours post fertilization (hpf), respectively), such as those stained using whole mount in situ hybridization (WISH), it is often desirable to remove the embryo from the yolk ball and to position it flat on a glass slide. However, performing a flat mount procedure can be tedious. Therefore, successful and efficient flat mount preparation is greatly facilitated through the visual demonstration of the dissection technique, and also helped by using reagents that assist in optimal tissue handling. Here, we provide our WISH protocol for one or two-color detection of gene expression in the zebrafish embryo, and demonstrate how the flat mounting procedure can be performed on this example of a stained fixed specimen. This flat mounting protocol is broadly applicable to the study of many embryonic structures that emerge during early zebrafish development, and can be implemented in conjunction with other staining methods performed on fixed embryo samples.

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is an excellent model for developmental biology research. During embryogenesis, zebrafish develop with a yolk mass, which presents three-dimensional challenges for sample observation and analysis. This protocol describes how to create two-dimensional flat mount preparations of whole mount in situ (WISH) stained zebrafish embryo specimens.

Preparazione di montaggio schermo per l&#39;osservazione e l&#39;analisi di zebrafish campioni macchiato da Whole Monte<em&gt; In situ</em&gt; Ibridazione

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model ...

In vivo Imaging dell'intero cervello delle larve di pesce zebra

In Vivo Whole-Brain Imaging of Zebrafish Larvae Using Three-Dimensional Fluorescence Microscopy

As a vertebrate model animal, larval zebrafish are widely used in neuroscience and provide a unique opportunity to monitor whole-brain activity at the cellular resolution. Here, we provide an optimized protocol for performing whole-brain imaging of larval zebrafish using three-dimensional fluorescence microscopy, including sample preparation and immobilization, sample embedding, image acquisition, and visualization after imaging. The current protocol enables in vivo imaging of the structure and neuronal activity of a larval zebrafish brain at a cellular resolution for over 1 h using confocal microscopy and custom-designed fluorescence microscopy. The critical steps in the protocol are also discussed, including sample mounting and positioning, preventing bubble formation and dust in the agarose gel, and avoiding motion in images caused by incomplete solidification of the agarose gel and paralyzation of the fish. The protocol has been validated and confirmed in multiple settings. This protocol can be easily adapted for imaging other organs of a larval zebrafish.

Autore in primo piano: Imaging in vivo dell'intero cervello di larve di pesce zebra mediante microscopia a fluorescenza tridimensionale  

Presented here is a protocol for in vivo whole-brain imaging of larval zebrafish using three-dimensional fluorescence microscopy. The experimental procedure includes sample preparation, image acquisition, and visualization.

In vivo Imaging dell'intero cervello delle larve di zebrafish mediante microscopia a fluorescenza tridimensionale

As a vertebrate model animal, larval zebrafish are widely used in neuroscience and provide a unique opportunity to monitor whole-brain activity at the ...

Analisi di apoptosi in embrioni di zebrafish da Whole-mount immunofluorescenza per rilevare caspasi 3 attivata

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Osservando divisione mitotica e Dynamics in un embrione vivo Zebrafish

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Utilizzo dell'immunofluorescenza per rilevare il danno al DNA indotto da PM_2,5nei cuori embrionali di zebrafish

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Un modello di embrione di Zebrafish per nel Vivo visualizzazione e analisi videomicroscopia del biomateriale-collegata dello stafilococco aureo infezione

Singolo Destino Mapping di celle in Zebrafish

Preparazione di montaggio schermo per l'osservazione e l'analisi di zebrafish campioni macchiato da Whole Monte

In vivo Imaging dell'intero cervello delle larve di zebrafish mediante microscopia a fluorescenza tridimensionale

In vivo Imaging dell'intero cervello delle larve di zebrafish mediante microscopia a fluorescenza tridimensionale