Quantifying Trophectoderm and Inner Cell Mass

Cells that were TE were distinguished from cells in the ICM based on nuclear labeling for CDX2 using procedures similar to those previously published [4 (link), 25 (link), 44 (link), 45 (link)]. The primary antibody was ready-to-use mouse monoclonal antibody against CDX2 (BioGenex, Fremont, CA, USA; reference AM392-5 M, lot AM3920917) For randomly-selected embryos, mouse 1gG (1 μg/mL) was used as a negative control. The second antibody was 1 μg/ml fluorescein isothiocyanate labeled goat anti-mouse IgG. Nuclei were labeled with 1 μg/ml Hoechst 33342. Fluorescence was observed with a Zeiss Axioplan 2 epifluorescence microscope (Zeiss, Gottingen, Germany) with a 40x objective and using Zeiss filter sets 02 (blue) and 03 (green). Digital images were acquired using AxioVision software (Zeiss) and a high-resolution black and white Zeiss AxioCam MRm digital camera. Total cell number was determined by counting nuclei labeled with Hoescht 33,342, number of TE cells was determined by counting nuclei positive for CDX2 and number of ICM cells was determined by subtracting number of TE cells from total cell number.