R3 34

Biotinylated lipid-shelled decafluorobutane microbubbles were prepared by sonication of a gas-saturated aqueous suspension of distearoylphosphatidylcholine (2 mg/mL), polyoxyethylene-40-stearate (1 mg/mL), and distearoylphosphatidylethanolamine-PEG(2000)biotin (0.4 mg/mL). Conjugation of biotinylated ligand to the microbubble surface was performed with biotin-streptavidin bridging as previously described.(15 (link)) Ligands used for targeting were: dimeric murine recombinant A1 domain of VWF A1 (mature VWF amino acids 445 to 716) for targeting platelet GPIbα for MB_Plt, a cell-derived peptide representing the N-terminal 300 amino acids of GPIbα for MB_VWF,(7 (link)) and monoclonal antibodies against the extracellular domain of either P-selectin (RB40.34, BD Biosciences), or VCAM-1 (clone 429, BD Biosciences) for MB_P and MB_V, respectively. Control MB were prepared with isotype control antibody (R3-34, BD Biosciences). Microbubble concentrations and size distributions were measured by electrozone sensing (Multisizer III, Beckman Coulter).

ACLP^+/− pregnant females were injected intravenously with a neutralizing antibody against IL-5, (TRFK5; BD Pharmingen) or an isotype control (R3-34; BD Pharmingen) for five consecutive days, using a dose of 100 μγ on E13.5-E14.5 and 150 μγ on E15.5-E17.5. The amount of antibody was calculated to achieve a fetal dose of 10mg/kg (the same dose that is typically used in an adult mouse (13 (link))) and an estimated maternal-fetal transmission of 2-3% (14 (link)). Fetal intestines and liver were analyzed on E18.5. In the case of fetal intervention, fetuses were injected with the same antibodies on E13.5 using a fetal intrahepatic injection as previously described (15 -17 (link)). Briefly, mothers were anesthetized and the uterus was exposed by performing a laparotomy. The fetuses were injected individually, through the translucent intact uterus, with the neutralizing antibody; controls were injected with the isotype control antibody or PBS in a volume of 5 μl, using pulled glass micropipettes. The laparotomy was closed in layers and the fetuses were analyzed on E18.5.

Biotinylated lipid-shelled decafluorobutane microbubbles (MB) were prepared by sonication of a gas-saturated aqueous suspension of distearoylphosphatidylcholine (2 mg/mL), polyoxyethylene-40-stearate (1 mg/mL), and distearoylphosphatidylethanolamine-PEG (2000) biotin (0.4 mg/mL) (Avanti Polar Lipids). Surface conjugation of biotinylated ligands was performed as previously described using a streptavidin bridge.^{14 (link)} Microbubbles targeted to platelet GPIbα (MB-A1) were prepared by surface conjugation of dimeric recombinant murine VWF A1 domain (mature VWF amino acids 445 to 716).^{9 (link)} Microbubbles targeted to VWF (MB-GC300) were prepared using a cell-derived biotinylated peptide representing the N-terminal 300 amino acids of GPIbα. Control non-targeted microbubbles (MB) were prepared using either human VWF A1 domain with a loss-of-function mutation (G561S) or a non-specific non-binding control monoclonal antibody (mAb) (R3-34, BD Biosciences) as appropriate. Microbubble concentrations and size distributions were measured by electrozone sensing (Multisizer III, Beckman Coulter).