Analysis of Cell Polarity in Tissue Sections

Frozen tissue sections were immunostained with anti-GM130 (mouse monoclonal), anti-CXCR4 (clone UMB2) and anti-PECAM-1 antibodies, as described in ‘General Immunohistochemistry and Quantification’, with the modification that Mouse on Mouse (M.O.M®) Basic Kit (Vector, BMK-2202) reagents were used for blocking sections and dilution of antibodies. Stained sections were imaged on an inverted Zeiss LSM 710 confocal using the 20x Plan Apochromat dry objective. Confocal stacks of sections were exported to FIJI and analyzed to assign orientation to individual CXCR4-positive cells. Positioning of the Golgi and nucleus was used to identify the anterior of cells and their orientation with respect to the nearest endocardial surface. Cell orientation was assigned depending on the angle between a straight line bisecting the Golgi/nucleus of each cell, and the plane of the endocardium, according to the 180 degree sectors illustrated in Figure 3S. The number of cells in each direction category was calculated as a proportion of the total number of CXCR4 positive cells per section analyzed.

For Cxcl12 nulls and controls, 3-6 sections from 4 embryos of each genotype were analyzed;

for Cxcr7 nulls and controls, 4-6 sections from 3 embryos per genotype were analyze.