Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

Summary

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.

Abstract

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.).

Introduction

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 preimplantatio....

Protocol

Ethics statement: All animal work, including husbandry, breeding and sacrifice was approved by Memorial Sloan Kettering Cancer Center'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.

1. Mate a virgin female mouse with a fertile stud male of the desired genotypes.
   Note: If setting up natural matings, selecting females in the estrus phase of the estral cycle increases the chances of cop....

Results

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.

The quality and reliability of t.......

Discussion

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 .......

Materials

List of materials used in this article

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