Intraductal Injection of MCF7 Cells in NSG Mice

Three 8 to 12-week-old NSG female mice were anesthetized by intraperitoneal injection with 10 mg/kg xylazine and 75 mg/kg ketamine (Graeub). To prevent eyes from drying while anesthetised, ophthalmic ointment (Viscotears) was applied to the eyes of mice. After shaving and disinfecting the area around the nipple, the intraductal injection was performed by injecting 500,000 MCF7 cells (resuspended in 10 µL of PBS) into the cleaved 3rd and 4th teats with a blunt end Hamilton syringe (HAMI80508, specifications: 50 µL 705 N, gauge 30/13 mm/pst3), as previously described^{46 (link)}. Paracetamol was added to the drinking water (200 to 300 mg/kg, i.e. 500 mg/250 mL of drinking water) from 1 day prior to the procedure until for 3 days after the intraductal injection. MCF7 cells were grown intraductally for 12 weeks to form mammary tumours. At the end of the period, mice were euthanized by CO₂ inhalation. Engrafted mammary glands were harvested and fixed in 4% PFA for 2 h, prior to dehydration and paraffin embedding.