Prohance

Brains left within the skull were prepared for MRI using the following method. Mice were anesthetized and intracardially perfused at a rate of 1 ml min⁻¹, with 30 ml of 0.1 M PBS containing 10 U/100 ml heparin (Sigma-Aldrich) and 2 mM ProHance (gadolinium contrast agent, Bracco Diagnostics) followed by 30 ml of 4% paraformaldehyde (PFA) and 2 mM ProHance. After perfusion, the mice were decapitated, and the skin and the lower jaw were removed. Brains in skulls were post-fixed in 4% PFA and 2 mM ProHance at 4 °C overnight and then kept in 0.1 M PBS containing 2 mM ProHance and 0.02% sodium azide for at least 1 month before MRI scanning^{61 (link),62 (link)}.

On PND 35-36, brains were flushed via transcardial perfusion (flow rate of 2 mL/min) with 50 mL phosphate-buffered saline (PBS) containing 10 U/mL heparin and 2 mM ProHance (a gadolinium-based contrast agent; Bracco Diagnostics Inc.), fixed with 50 mL 4% paraformaldehyde (PFA) in PBS containing 2 mM ProHance, and collected for neuroimaging following previously published protocols [36 (link)]. Following perfusion, brains were incubated in the 4% PFA solution for 24 h at 4 °C then transferred to a storage PBS solution containing 0.02% sodium azide. Brains were incubated in the storage solution at 4 °C for at least 1 month prior to scanning. Images were acquired and analyzed following a protocol previously described [33 (link), 37 (link)]. Multiple comparisons were controlled for using the false discovery rate (FDR) with the significance level for the FDR-adjusted p value (q) set at q < 0.05 [38 (link)].

Mice were anesthetized and intracardially perfused as previously described [44 (link)]. Briefly, following transcardial perfusion with heparin, 4% paraformaldehyde and 2 mM ProHance (Bracco Diagnostics), heads were decapitated and the skin, lower jaw, ears, and the cartilaginous nose tip were removed. The remaining skull containing the brain was soaked overnight in 4% paraformaldehyde/2 mM ProHance and then transferred to PBS, 0.02% sodium azide and 2 mM ProHance for at least 30 days prior to magnetic resonance imaging (MRI) acquisition [45 (link)].

MRI was performed at our institution using a standard and consistent clinical breast MRI protocol. Women were imaged in the prone position by a 1.5 T scanner (GE Signa EXCITE, GE Health, Nutley, NJ, USA) using a dedicated 7-channel surface array breast coil (InVivo, Gainesville, FL, USA). Imaging parameters were: matrix 512 × 512; slice thickness 2 mm; field of view 28–34 cm, flip angle 10°, repetition time (TR) 5.68 msec, echo time (TE) 2.736 msec. Bolus injection of the contrast agent, ProHance (Bracco Diagnostics, Princeton, NJ, USA), at 0.1 mmol/kg, 3 cc/sec was followed by a 20-cc saline flush. The first post-contrast sequence acquisition was centered at 90 seconds after contrast agent injection. A pre-contrast sequence and three sequential time point post-contrast sequences were acquired in the axial view for bilateral breasts, where each sequence took approximately 3 minutes to complete, depending on field of view sizes selected to cover the breasts. Three subtraction sequences (SUB1, SUB2, and SUB3) were generated by subtracting the pre-contrast sequence from each of the three post-contrast sequences, respectively, as part of routine post-processing with CADstream (Merge Healthcare Inc., Chicago, IL, USA).