*4.7. Morphometric Analyses*

Morphometric measurements of protein expression and blood vessel densities were determined on tissue immunostained for endothelial cell marker, CD31, mural cell markers NG2, PDGFRβ and SMA, and Notch ligands Jagged1 or Dll4. The mesometrial region and anti-mesometrial region of each implantation site were identified and further divided into mesometrial pole, defined as the area extending from the internal border of the myometrium to the uterine lumen, and the central region, defined as the areas of the implantation site lateral to and flanking the embryo and implantation chamber (Figure 1A,B). For blood vessel analysis, CD31 signal density was measured in 5 random 0.025 mm<sup>2</sup> areas of the decidua in these three regions using ImageJ Software Version 2.0.0 (mesometrial pole, central region and anti-mesometrial region) [7]. Expression of Jag1 and Dll4 was determined by measurements of the signal densities and divided by CD31<sup>+</sup> signal density or NG2+ or PDGFRβ <sup>+</sup> signal density in 5 random 0.025 mm<sup>2</sup> areas of the decidua. To quantify expression of N1ICD with respect to CD31, number of N1ICD<sup>+</sup> nuclei was determined for 5 random 0.037 mm<sup>2</sup> areas of anti-mesometrial decidua. The CD31 signal density was determined for each of these areas, and number of nuclei per square micrometer of CD31<sup>+</sup> ECs was calculated. For analysis of VEGFR2 in CD31<sup>+</sup> ECs, signal densities were evaluated in adjacent sections immunostained with VEGFR2 or CD31. The signal density was measured in 5 random 0.025mm<sup>2</sup> areas of the decidua in like-regions for both VEGFR2 and CD31 and signal densities of VEGFR2 were divided by that of CD31<sup>+</sup> cells. Sections from 2–3 implantation sites per each mouse were examined and were used for the analyses.
