Immunofluorescence analysis of GFP expression during mouse development. (A-F) Whole-mount staining of yolk sacs from CD68-GFP (A,C-F) and WT (B) embryos at E12.5 to detect the presence of GFP⁺ cells (monocytes and macrophages) by immunofluorescence with an anti-GFP monoclonal antibody (green). Costaining with PECAM-1 (red), an endothelial cell marker, shows association of GFP⁺ cells with vessels (arrowheads in D,F). Nuclei are stained with DAPI (blue). (G-O) Frontal cryosections from CD68-GFP mouse embryos at E14.5. Immunofluorescence staining shows GFP⁺ cells (green) in the liver, heart, and thymus (G,J,M). F4/80⁺ cells (red) are abundant in the liver and thymus, but fewer F4/80 macrophages are seen in the heart at this stage in development (H,K,N). Arrowheads in panels L and O mark cells that are both GFP and F4/80 positive. Nuclei are stained with DAPI (blue).