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ARS Home » Research » Publications at this Location » Publication #118858
Title: A SIMPLE, RAPID PROCEDURE FOR DIFFERENTIAL STAINING OF TROPHECTODERM AND INNER CELL MASS CELLS.

Author
item Wells, Kevin
item HILL, J - CORNELL UNIVERSITY

Submitted to: Theriogenology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/26/2001
Publication Date: N/A
Citation: N/A

Interpretive Summary: The early mammalian embryo is composed of two cell layers, the trophectoder (TE) and and the inner cell mass (ICM). The number and proportion of cells that populate the ICM and TE can be useful determinations for evaluation of treatment effects. A simple and rapid protocol is described as an alternative to standard procedures to allow differential nuclear staining of ICM and TE. The basic protocol requires staining of all cells with Hoechst 33342 (blue) to equilibrium. Embryos are then exposed to a non- ionic detergent (0.04% TritonX-100) for 1-5 minutes to permeablize the TE, depending on species and condition of the embryo. After detergent exposure, the embryos are stained for greater than 4 minutes in propidium iodide (red) which penetrates only the detergent damaged TE layer which encapsulates the embryo. When illuminated with UV light, the ICM is fluorescent blue and the TE is fluorescent red. This basic protocol has been adapted to stain embryos from cattle, mice, and pigs and should be adaptable to most other mammalian species. Scientist that need to evaluate development of mammalian embryo will find this procedure useful.

Technical Abstract: As evaluation criteria, the number and proportion of cells that populate the inner cell mass (ICM) and the trophectoderm layer (TE) can be useful determinations. A simple and rapid protocol is described as an alternative to standard procedures to allow differential nuclear staining of ICM and TE. The basic protocol requires staining with Hoechst 33342 (Ho) to equilibrium. Embryos are then exposed to 0.04% TritonX-100 for 1-5 minutes to permeablize the TE, depending on species and presence of the zona pellucida. After TritonX-100 exposure, the embryos are stained for greater than 4 minutes in propidium iodide (PI). Stained embryos can be visualized with epifluorescence using a standard DAPI/Ho wide pass filter. The red PI fluorescence is sufficiently bright with ultraviolet excitation to be seen with the blue filter. This basic protocol has been adapted to stain bovine, murine, and porcine embryos and should be adaptable to most species.