Name: quantitative analysis of protein expression to study lineage specification in mouse preimplantation embryos
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The authors would like to thank Stefano Di Talia, Alberto Puliafito, Venkatraman Seshan and Panagiotis Xenopoulos, for input on data handling, analysis and representation, Berenika Plusa for assistance in the design of the immunofluorescence protocol and antibody testing and members of the Hadjantonakis lab for comments on the manuscript and on the development of this protocol. Work in our lab is supported by the National Institutes of Health R01-HD052115 and R01-DK084391, and by NYSTEM N13G-236.

Ethics statement: All animal work, including husbandry, breeding and sacrifice was approved by Memorial Sloan Kettering Cancer Center&#39;s Institutional Animal Care and Use Committee (IACUC), protocol #03-12-017.
1. Embryo Collection
Note: All animal work must have been approved by institutional and local authorities and conform to local and institutional rules.
<ol>
	<li>Mate a virgin female mouse with a fertile stud male of the desired genotype...

<ol>
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The present protocol describes a method to perform a quantitative analysis of whole-mount immunofluorescence on preimplantation stage mouse embryos. A robust immunofluorescence protocol 22 is followed by high-resolution, whole-mount confocal imaging and by image segmentation using a tailored piece of software 4. While the choice of immunofluorescence protocol is not critical, we find the one presented here 22 to be fast, reliable and to provide robust signal for many of the antibodies we ...

The mouse preimplantation embryo is a paradigm to study the emergence and maintenance of pluripotency in vivo, as well as a model for the study of cell fate specification and de novo epithelialization in mammals. The preimplantation stages of mammalian development are dedicated to the establishment of the three cell lineages that make up the blastocyst, namely the pluripotent epiblast - which gives rise to most somatic tissues and germ cells - and two extraembryonic lineages, the trophectoderm (TE) and the primitive endoderm (PrE) (Figure 1A) 1,2. This protocol describes the procedures to (1) harvest and fix preimplantation stage mouse embryos, (2) perform immunofluorescence to label proteins of interest, (3) carry out whole-mount imaging using a confocal microscope with z-sectioning capabilities and (4) perform nuclear segmentation of confocal images and subsequent quantitative image analyses. This pipeline allows the unbiased measurement of protein levels for the assignment of cell identities to characterize subpopulations of cells in situ. This protocol can be carried out in as little as 3 - 4 days for a single litter (generally up to 10 mouse embryos), from embryo collection to the data analysis (Figure 1B). The simultaneous analysis of several litters would increase the imaging and data analysis time burden, thus extending the overall length of the protocol.
The preimplantation stage mouse embryo is an experimentally tractable system which, given its small size and stereotypical morphology 3, is well suited for in toto imaging of cellular processes with single-cell resolution. To carry out an unbiased, systems-level analysis of a statistically relevant number of embryos, an automated, quantitative analysis pipeline is desirable. However, due to the high nuclear density of the inner cell mass (ICM) of the blastocyst (Figure 1A, 2D), conventional image segmentation platforms fail to provide sufficient accuracy to establish an automated or semi-automated workflow. On the other hand, manual segmentation, while accurate, does not allow the processing of large cohorts of cells and embryos, nor is it suitable for a reproducible, unbiased determination of cell identities - which is especially critical when studying developmental stages where patterns of marker expression have not fully resolved (e.g., do not exhibit a binary distribution across a population). We have recently developed and validated an image segmentation method tailored for mouse preimplantation stage embryos and for mouse embryonic stem cells (ESCs) that achieves high accuracy, while requiring minimal user input 4-8.
The analysis pipeline presented here revolves around the MATLAB-based image segmentation tool Modular Interactive Nuclear Segmentation (MINS) 4. MINS performs unsupervised nuclear segmentation on large batches of confocal Z-stacks after the user has established a minimal number of image properties, using a graphical user interface (GUI) (Table 1) 4. This pipeline has proven efficient for the generation of high throughput data on protein expression and cell localization in both wild type, experimentally treated and genetically modified embryos and ESCs 5-7. In the present protocol, we describe the application of MINS to the segmentation of preimplantation-stage embryo images. For examples of MINS performance on ESCs please refer to 4,7. The automated nuclear segmentation step significantly reduces the time burden of the cell identification process, whereas the spatial and fluorescence intensity measurements allow an unbiased determination of cell identities and the generation of three-dimensional maps of gene expression domains and cell position in the embryo (Figure 1C). Moreover, the scalability of this workflow makes it applicable to the analysis of individual litters through large cohorts of experimentally treated embryos, or embryos of different genetic backgrounds 5,6. MINS is freely available at&#160; http://katlab-tools.org (the software requires a MATLAB license).
No approach developed to date allows the generation of such in-depth data on protein expression and cell localization in mouse preimplantation embryos. All attempts thus far at quantifying these types of data have been restricted to the manual determination and quantitation of cell numbers for different populations in the embryo (either entirely manually, or software-assisted) 9-19. This approach (incorporating MINS software) has been tailored for and tested on mouse preimplantation embryos and ESCs; nevertheless its performance on other systems with high nuclear density, although yet untested, is expected to be equivalent.

<table border="0" cellpadding="0" cellspacing="0" width="1365">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Embryo collection</td>
			<td style="width:157px;"></td>
			<td style="width:143px;"></td>
			<td style="width:743px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blunt probe for plug checking</td>
			<td>Roboz</td>
			<td>RS-9580</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forceps</td>
			<td>Roboz</td>
			<td>RS-4978</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surgery scissors</td>
			<td>Roboz</td>
			<td>RS-5910</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass Pasteur pipettes</td>
			<td>Fisher</td>
			<td>13-678-20C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pre-assembled aspirator tube &#38; mouthpiece</td>
			<td>Sigma</td>
			<td>A5177</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Longer rubber tubing</td>
			<td>Fisher</td>
			<td>14-178-2AA&#160;&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">M2</td>
			<td>Millipore</td>
			<td>MR-015-D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FHM</td>
			<td>Millipore</td>
			<td>MR-024-D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acid Tyrode&#39;s solution</td>
			<td>Millipore</td>
			<td>MR-004-D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin/Streptomycin</td>
			<td>Gibco</td>
			<td>15140</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine Serum Albumin&#160;</td>
			<td>Sigma</td>
			<td>A9647</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4-well plates</td>
			<td>Nunc/Thermo-Fisher</td>
			<td>12-566-300&#160;&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immunofluorescence</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96-well U-bottom plates</td>
			<td>Fisher</td>
			<td>14-245-73</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycine</td>
			<td>Sigma</td>
			<td>G7403</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Horse serum</td>
			<td>Sigma</td>
			<td>H0146</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Primary antibodies</td>
			<td></td>
			<td></td>
			<td>Concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CDX2</td>
			<td>Biogenex</td>
			<td>AM392-5M</td>
			<td>1:200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GATA6</td>
			<td>R&#38;D</td>
			<td>AF1700</td>
			<td>1:100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GATA4</td>
			<td>Santa Cruz</td>
			<td>sc-9053</td>
			<td>1:100, 10 min fixation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GATA4</td>
			<td>Santa Cruz</td>
			<td>sc-1237</td>
			<td>1:100, overnight fixation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SOX17</td>
			<td>R&#38;D</td>
			<td>AF1924</td>
			<td>1:100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nanog</td>
			<td>ReproCELL</td>
			<td>RCAB0002P-F</td>
			<td>1:500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">OCT4</td>
			<td>Santa Cruz</td>
			<td>sc-5279</td>
			<td>1:100, 10 min to overnight fixation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DAB2</td>
			<td>BD</td>
			<td>BD-610464</td>
			<td>1:200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Secondary antibodies</td>
			<td>Life Technologies</td>
			<td>Various</td>
			<td>1:500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoechst 33342</td>
			<td>Life Technologies</td>
			<td>H3570</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Imaging</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">35 mm glass-bottom dishes</td>
			<td>MatTek</td>
			<td>P35G-1.5-14-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Segmentation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Computer running 64-bit Windows OS</td>
			<td></td>
			<td>n/a</td>
			<td>Verify minimal system requirements at http://katlab-tools.org and in Lou et al., (2014) Stem Cell Reports</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MATLAB (software)</td>
			<td>Mathworks</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MINS (software)</td>
			<td>Free</td>
			<td>n/a</td>
			<td>http://katlab-tools.org</td>
		</tr>
	</tbody>
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quantitative analysis of protein expression to study lineage specification in mouse preimplantation embryos

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for embryo collection, immunofluorescence, imaging on a confocal microscope, and image segmentation and analysis are described. This method allows quantitation of the expression of multiple nuclear markers and the spatial (XYZ) coordinates of all cells in the embryo. It takes advantage of MINS, an image segmentation software tool specifically developed for the analysis of confocal images of preimplantation embryos and embryonic stem cell (ESC) colonies. MINS carries out unsupervised nuclear segmentation across the X, Y and Z dimensions, and produces information on cell position in three-dimensional space, as well as nuclear fluorescence levels for all channels with minimal user input. While this protocol has been optimized for the analysis of images of preimplantation stage mouse embryos, it can easily be adapted to the analysis of any other samples exhibiting a good signal-to-noise ratio and where high nuclear density poses a hurdle to image segmentation (e.g., expression analysis of embryonic stem cell (ESC) colonies, differentiating cells in culture, embryos of other species or stages, etc.).

To facilitate data interpretation and presentation, care should be taken not to damage the embryos during collection and manipulation, so that all cells and their relative position can be analyzed. Figure 2A - D shows examples of intact blastocysts at different stages with an expanded cavity. Should damage occur, extra care should be taken when analyzing and interpreting results.
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Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

This protocol presents a method to perform quantitative, single-cell in situ analysis of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for collection of blastocysts, whole-mount immunofluorescent detection of proteins, imaging of samples on a confocal microscope, and nuclear segmentation and image analysis are described.

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse ...

The overall goal of this protocol is to quantify protein levels in situ in mouse preimplantation embryos. So this method allows us to perform high-throughput analysis of confocal microscopy images with single-cell resolution in a semi-automated way. This method can be used to quantify protein concentrations on confocal images where there is high nuclear density and when one wants to study heterogeneities within cell populations.To begin, use an open flame on a glass pasteur pipette to draw a capillary end at the tip. Gently rub the far end of the pipette against the capillary, to break it while creating a blunt end. After sacrificing a pregnant female mouse on the desired embryonic day of development, expose the abdominal viscera according to the text protocol and locate the uterus.Hold the cervical end, cut through the cervix, and gently pull up to stretch both uterine horns. Trim the fat from it, taking care not to pierce the uterine wall. Cut above the oviducts, below the ovaries, to release the entire uterus, and place on a petri dish.Next, cover the uterus with PBS, and separate both uterine horns by cutting through the proximal end on both sides of the cervix. Then separate each oviduct from the uterus by cutting below the isthmus that connects them. Under a dissection microscope, use a one mL syringe with a hypodermic needle to flush the blastocysts out of the uterus and into manipulation medium, by forcing 0.5-1 mL of medium through each horn from the cervical opening.Allow up to one to two minutes for blastocysts to sink to the bottom of the dish after flushing. Then locate the blastocysts, and using the mouth-controlled glass pasteur pipette with a capillary end, collect the blastocysts and transfer to a fresh drop of medium. After rinsing the blastocysts, remove the zona pellucida by using Acidic Tyrode's Solution to briefly wash the embryos.As soon as the zona is no longer visible, transfer the blastocysts back to manipulation medium. Wash the blastocysts in room temperature PBS, and fix them by transferring to 4%PFA in PBS for 10 minutes at room temperature. After fixation, transfer the blastocysts back to PBS for storage at four degrees Celsius.After carrying out imunoflourescence according to the text protocol, use a fine mouth pipette to make microdrops of PBS or nuclear stain solution on the glass surface of a 35mm glass-bottom dish, and cover them with mineral oil. Place the embryos in the microdrops and arrange in a consistent manner, preferably with the ICM cavity access parallel to the glass surface, then set up the dish on the microscope holder. To carry out confocal imagining, use the lowest laser power that provides a strong signal-to-noise ratio without bleaching the fluorophores.Adjust the gain and off set to obtain the widest dynamic range without overexposing the sample. Capturing most of or the entire grayscale range will facilitate the detection of small differences in intensity between images. Follow the additional details outlined in the text protocol to image embryos throughout the entire Z axis.Follow the graphic user interface of the MATLAB-based segmentation tool Modular Interactive Nuclear Segmentation, or MINS, to load a confocal image or an entire Z stack of raw data generated by the microscope. To view the outcome of each step, click the corresponding View"button. A yellow tag above the button indicates operation in process.A green tag indicates operation completed. Assess the outcome of the detection step before proceeding to segmentation. If it is not satisfactory, modify the detection parameters and rerun it.From the Batch Mode"run menu, click Add Files"and load all files to be processed at once. Click Start Batch Mode Run"and allow time for the software to process the files. Segmentation output will be saved in the same directory as the original files.Identify false positives such as apoptotic vesicles or other elements that are not intact nuclei but which may have been identified as such by MINS. Delete the corresponding records for false positives from the starstatistics. csv file.Preserve the original starstatistics. csv file for future reference and edit only a copy of it. If a nucleus has been over segmented and presented as two or more nuclei, either merge the records by averaging their intensity level, or keep one of the records for that cell and discard the rest.To resolve under segmentation, identify events where MINS has failed to detect the border between two or more nuclei and segmented them as a single one, or where it has failed to detect a nucleus altogether. Use ImageJ to measure the average gray level for each channel in the under or unsegmented cells. Next, find a medial section of the under or unsegmented nucleus on the DNA channel, using the freehand selection tool, outline the perimeter of the under or unsegmented nucleus.Then, press Control M"or go to the Analyze"menu and select Measure"This will record the mean gray value for the area outlined and will display it on a new window. Using the same outlined area just selected in the DNA channel, repeat the measurement of each of the fluorescence channels of interest. The results will be upended to the previous one on the measurements results window.Then use the measurements obtained to replace the erroneous records in the starstatistics. csv file. If the nucleus has been undersegmented duplicate its row, assign different cell IDs to each new cell, and introduce the values obtained in ImageJ under the corresponding column.In this case, both cells will share spacial coordinates. When mitotic nuclei are to be considered separately in the analysis, manually score them and add the information to the data file. Refer to the text protocol for additional image processing instructions.This figure shows examples of intact blastocysts at different stages with an expanded cavity. Shown here are examples of good antibodies for a number of nuclear proteins, including CDX2, GATA4, GATA6, NANOG, and OCT4. This sample was labeled with the cytoplasmic protein DAB2 that gives a high signal-to-noise ratio.In this panel, examples of a bad staining for GATA4 with a low signal-to-noise ratio are shown, where the samples were only fixed for 10 minutes. This particular anti-GATA4 antibody requires overnight fixation to provide a strong signal. These embryos were imaged with a 40x oil immersion objective with an NA of 1.30 and a 0.21 mm working distance The middle panels show magnifications of the ICMs or individual nuclei and the border between them can be distinguished.The bottom panels illustrate how the more advanced the embryo, the higher the nuclear density, and thus the greater the chance of segmentation errors. These images show errors that MINS can commit, such as detecting apoptotic nuclei as live cells, over segmentation, or under segmentation. This sequence of Z slices reveals an under segmentation event, where two cells have been identified as one.Once mastered, this protocol can be carried out in three to four days from beginning to end, with a time burden of two to four hours per day. While performing this procedure, it is important to remember to be very consistent with experiment conditions, that is immunofluorescent protocols and imaging parameters. After its development, this technique paved the way for researchers in the field of mouse developmental biology to perform in situ quantitative analysis of gene and protein expression in single cells.After watching this video, you shall have a good understanding of how to acquire image data that is appropriate for quantitative in situ expression analysis.

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