Name: quantitative whole-mount immunofluorescence analysis of cardiac progenitor populations in mouse embryos
Uploaded: 2017-10-12T15:00:00
Description: Watch this Scientific Journal Video about Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos at JoVE.com

This work was funded by NIH/NHLBI R56 HL128646 and the Mindich Child Health and Development Institute (MCHDI) at ISMMS (to N.D.). E.B. is supported by an NIH/NIDCR Traineeship T32 HD075735.Microscopy and image analysis was performed at the Microscopy Core at the Icahn School of Medicine at Mount Sinai, which is supported in part by the Tisch Cancer Institute at Mount Sinai P30 CA196521 &#8211; Cancer Center Support Grant.

All methods described here have been approved by the Institutional Animal Care and Use Committee at the Icahn School of Medicine at Mount Sinai.
1. Harvesting and Processing Cardiac Crescent Stage Embryos
<ol>
	<li>Mate a fertile female mouse with a fertile stud male.</li>
	<li>Check for the presence of a vaginal copulation plug using a blunt probe or forceps. Noon on the day of plug detection is considered embryonic day (E) 0.5 (See Figure 1A for full timeline)...

<ol>
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The protocol above describes a strategy for obtaining quantitative data from high quality whole mount immunofluorescence images of post-implantation mouse embryos. When performed correctly, the 3D volumetric data generated through these steps can be used for comparative and intersectional analysis of multiple domains within the embryo. The surface signal masking approach described is of particular use when investigating novel cell populations in comparison with well-established reference structures.
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The study of organogenesis has long relied on the observation of morphogenetic events in the developing embryo. These studies frequently rely on the use of fluorescent dyes or lineage tracing reporters in combination with labeling of defined reference populations.1 By comparing relative positions of these labels, information can be gleaned on the origin, movement, or ultimate contribution of a population of interest. Transplantation and fate mapping experiments use either morphological landmarks or injection of dyes into non-motile lineages to define the starting point of the cells of interest, which are then examined for contribution to the developed embryo.2,3,4,5 Genetic lineage-tracing experiments use the same concept with well-defined reporter alleles that are used to label cell populations without experimental manipulation. Key to these approaches is the ability to determine, with high spatial resolution, the locations of the experimental and reference labels. These approaches have yielded outstanding progress in pre-implantation development and explant organogenesis studies.6,7,8,9
The developmental events that underlie heart morphogenesis have been increasingly well described in recent years.10 One of the major discoveries in this area of research is the description of a number of progenitor populations that can be distinguished by expression of unique markers.11 These populations include the First and Second Heart Fields (FHF and SHF), which are present within the cardiac crescent at the anterior side of the embryo at embryonic day (E) 8.25 of mouse development.12 These populations are frequently examined through a combination of wide-field microscopy, which provides tissue-level information, and serial sectioning with immunofluorescence assays, which offers high cellular resolution but only two-dimensional spatial information.13 Thus, while these studies have greatly advanced our understanding of heart development, the available methods have limited in depth quantitative analysis of morphogenesis during these stages, creating the need for approaches to examine the organization of these populations on a whole-organism level.
The recent advances in both confocal microscopy and 3D image analysis allow for high-resolution and high-throughput algorithmic reconstructions of cells and structures in situ with relative ease, thus paving the way for detailed studies of complex cellular structures.14 With the increase of computational power and the development of big-data managing algorithms, both necessary to handle the exponential increase of the size of imaging data-sets, analyses can now be fully automated.15 Automated analysis of imaging data-sets has the benefit of being unbiased, but it is only as reliable as the quality of the input dataset; it is imperative, then, that best-practices are used during acquisition and image pre-processing to ensure the highest quality, unbiased analysis.16 Protocols can be completely automated and shared for reproducibility, and the algorithms used by proprietary software are readily available through libraries to be used by scientists who have familiarity with modern proprietary or open-source developer tools.17
The following protocol explains the necessary steps to perform such analysis on one well defined model of organogenesis, the formation of the cardiac crescent during heart development. Specifically, this protocol describes how to (1) harvest and dissect cardiac crescent stage embryos, (2) perform whole mount immunofluorescence for reference (Nkx2-5) and experimental (Foxa2Cre:YFP18,19) markers, (3) prepare and image the embryos using confocal microscopy, and finally (4) analyze and quantify the resulting images using advanced three-dimensional approaches. While the cardiac crescent is used as an example here, with appropriate modification, this protocol may be used for analysis of multiple lineages in gastrula to early somite stage embryos.

<table>
	<tbody>
		<tr>
			<td>Blunt probe</td>
			<td>Roboz</td>
			<td>RS-9580</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Roboz</td>
			<td>RS-8100</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>Roboz</td>
			<td>RS-5015</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection scissors</td>
			<td>Roboz</td>
			<td>RS-5912</td>
			<td></td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>Sigma Aldrich</td>
			<td>84510</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma Aldrich</td>
			<td>A8022</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton</td>
			<td>RPI</td>
			<td>A4490</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Nkx2-5</td>
			<td>Santa Cruz Biotech</td>
			<td>sc-8697</td>
			<td>Used at 1:100-1:500</td>
		</tr>
		<tr>
			<td>Chicken anti-GFP</td>
			<td>abcam</td>
			<td>ab13970</td>
			<td>Used at 1:500</td>
		</tr>
		<tr>
			<td>Rabbit anti-Islet1</td>
			<td>abcam</td>
			<td>ab109517</td>
			<td>Used at 1:100</td>
		</tr>
		<tr>
			<td>Rabbit anti-Hcn4</td>
			<td>Millipore</td>
			<td>AB5808</td>
			<td>Used at 1:100</td>
		</tr>
		<tr>
			<td>488 anti-chicken</td>
			<td>Jackson Immunoresearch</td>
			<td>703-546-155</td>
			<td>Reconstituted in water and stored at -20 &#176;C in final concentration of 50% glycerol. Used at 1:500.</td>
		</tr>
		<tr>
			<td>555 anti-goat</td>
			<td>Thermo Fisher Scientific</td>
			<td>A21432</td>
			<td>Used at 1:500</td>
		</tr>
		<tr>
			<td>647 anti-rabbit</td>
			<td>Jackson Immunoresearch</td>
			<td>711-606-152</td>
			<td>Reconstituted in water and stored at -20 &#176;C in final concentration of 50% glycerol. Used at 1:500.</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma Aldrich</td>
			<td>D9542</td>
			<td></td>
		</tr>
		<tr>
			<td>n-Propyl gallate</td>
			<td>Sigma Aldrich</td>
			<td>2370</td>
			<td>Stock solution is 20% w/v in DMSO. Working solution prepared by mixing 1 part 10x PBS with 9 parts 100% glycerol and slowly adding 0.1 part stock solution.</td>
		</tr>
		<tr>
			<td>Superfrost Plus microscopy slides</td>
			<td>VWR Scientific</td>
			<td>48311-703</td>
			<td></td>
		</tr>
		<tr>
			<td>22x22 mm coverslips</td>
			<td>VWR Scientific</td>
			<td>48366-227</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaris 8.4.1</td>
			<td>Bitplane</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

quantitative whole-mount immunofluorescence analysis of cardiac progenitor populations in mouse embryos

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development and organogenesis are two areas of research that have benefitted greatly from these advances, due to the high quality of data that can be obtained directly from imaging pre-implantation embryos or ex vivo organs. While pre-implantation embryos have yielded data with especially high spatial resolution, later stages have been less amenable to three-dimensional reconstruction. Obtaining high-quality 3D or volumetric data for known embryonic structures in combination with fate mapping or genetic lineage tracing will allow for a more comprehensive analysis of the morphogenetic events taking place during embryogenesis.
This protocol describes a whole-mount immunofluorescence approach that allows for the labeling, visualization, and quantification of progenitor cell populations within the developing cardiac crescent, a key structure formed during heart development. The approach is designed in such a way that both cell- and tissue-level information can be obtained. Using confocal microscopy and image processing, this protocol allows for three-dimensional spatial reconstruction of the cardiac crescent, thereby providing the ability to analyze the localization and organization of specific progenitor populations during this critical phase of heart development. Importantly, the use of reference antibodies allows for successive masking of the cardiac crescent and subsequent quantitative measurements of areas within the crescent. This protocol will not only enable a detailed examination of early heart development, but with adaptations should be applicable to most organ systems in the gastrula to early somite stage mouse embryo.

The quality of the final data and analysis depends greatly on (1) the integrity and morphology of the dissected embryos, (2) the use of high specificity antibodies, and (3) the proper setup of imaging parameters. Damaged embryos will confound the surface generation process and hinder quantitative analysis. Examples of properly dissected and staged embryos are shown in Figure 2B. The embryos can be further dissected by removing the yolk sac, to which antibodie...

Watch this Scientific Journal Video about Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos at JoVE.com

Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos

Here, we describe a protocol for whole mount immunofluorescence and image-based quantitative volumetric analysis of early stage mouse embryos. We present this technique as a powerful approach to qualitatively and quantitatively assess cardiac structures during development, and propose that it may be widely adaptable to other organ systems.

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development ...

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