For all experiments using microCT quantification of CCM lesion volume, brains were harvested and placed in 4% PFA/PBS. Brains remained in fixative until staining with nondestructive iodine contrast and subsequent microCT imaging performed as previously described (Girard et al., 2016 (link)).
Experiments where brains had observable hemorrhage (e.g., Krit1BECKO; ADAMTS5BEC-GOF) were subject to further analysis. For each mouse, the total blood volume (lesional + extra-lesional) was determine by microCT. Since extra-lesional bleeding cannot be distinguished from blood within caverns on microCT, histological analysis was used. With PECAM immunostaining, ECs were delineated on the largest sections of index lesions identified on microCT. Bleeding was identified on the respective histological images, and the area of extra-lesional blood (not confined by endothelium lining) and the lesional area (including any blood confined within endothelium lined caverns) were measured using the polygon area function of an Olympus DP2-SAL standalone connection kit attached to an Olympus DP22 digital camera mounted on top of an Olympus CX41 microscope.
Tissue processing, imaging, and volumetric quantification were performed in a blinded manner by investigators at the University of Chicago without prior knowledge of genotype or experimental details.
Experiments where brains had observable hemorrhage (e.g., Krit1BECKO; ADAMTS5BEC-GOF) were subject to further analysis. For each mouse, the total blood volume (lesional + extra-lesional) was determine by microCT. Since extra-lesional bleeding cannot be distinguished from blood within caverns on microCT, histological analysis was used. With PECAM immunostaining, ECs were delineated on the largest sections of index lesions identified on microCT. Bleeding was identified on the respective histological images, and the area of extra-lesional blood (not confined by endothelium lining) and the lesional area (including any blood confined within endothelium lined caverns) were measured using the polygon area function of an Olympus DP2-SAL standalone connection kit attached to an Olympus DP22 digital camera mounted on top of an Olympus CX41 microscope.
Tissue processing, imaging, and volumetric quantification were performed in a blinded manner by investigators at the University of Chicago without prior knowledge of genotype or experimental details.
