Bcl-xPf4Δ/Pf4ΔMcl-1Pf4Δ/Pf4Δ embryos exhibit hemorrhages and blood-filled lymphatic vessels. (A) Representative images from E12.5 wild-type, Bcl-xPf4Δ/Pf4ΔMcl-1+/+, Bcl-xPf4Δ/Pf4ΔMcl-1+/Pf4Δ, and Bcl-xPf4Δ/Pf4ΔMcl-1Pf4Δ/Pf4Δ embryos. Black arrowhead indicates areas of ectopic bleeding. Note, some embryos have been tailed for genotyping. (B) Immunofluorescent detection of blood vessels and lymphatic vessels using Abs directed against endomucin (green), Prox1 (red), and neuropilin2 (Nrp2; cyan). Blood-filled lymph sacs (iii-iv) and lymphatic vessels (v) are a feature of E12.5 Bcl-xPf4Δ/Pf4ΔMcl-1+/+ and Bcl-xPf4Δ/Pf4ΔMcl-1Pf4Δ/Pf4Δ, but not wild-type (i-ii) embryos. (vi)Hemorrhages were also observed in Bcl-xPf4Δ/Pf4ΔMcl-1Pf4Δ/Pf4Δ embryos. Z-stack images were obtained using a Bio-Rad Radiance 2100 confocal microscope equipped with 3 lasers (488-nm Argon-ion laser, HeNe 543-nm green laser, 637-nm red diode laser) attached to an Olympus IX70 inverted microscope with UPlanApo 10×/0.40 ∞/0.17 and UApo/340 20×/0.70W ∞/0.17 objectives. Image acquisition was performed at room temperature using LaserSharp 2000 software (Version 5.2; Bio-Rad Laboratories). Adobe Photoshop CS5 (Version 12.0; Adobe) was used for subsequent image processing. LS indicates lymph sac; CV, cardinal vein; LV, lymphatic vessels; and V, ventricle. White arrowheads indicate blood-filled lymph sacs/lymphatic vessels and white asterisks (*) indicate areas of hemorrhage. Scale bars: 120 μm (white), 80 μm (yellow), and 240 μm (blue).
