Staining and Imaging of Live Rabbit Embryos

INTRODUCTION

This protocol describes methods for supravital labeling of cellular proliferation and movement in rabbit embryos as well as for imaging of live cell movements. The techniques use localized labeling of epiblast cells with DiI by injection or by using comprehensive nuclear staining of all cell layers before and during early gastrulation. Because rabbit embryos implant relatively late during embryonic development, gastrulation-stage embryos can be isolated by flushing them from the uterus. Moreover, the embryonic disc is flat and translucent, providing a direct uncompromised overview of gastrulation while the whole blastocyst is protected by the zona pellucida. These properties allow examination of whole blastocysts either under the impact of changing environmental conditions (e.g., medium additives, oxygen concentrations) or following mechanical manipulation of the embryonic disc using a needle penetrating the zona pellucida. Further manipulation (e.g., transplantation) can be performed after removing the embryo from the zona and explantation of the embryonic disc. These techniques allow embryonic growth centers to be identified and single-cell movements to be recorded.