Mammary Gland Transplantation and Tumor Monitoring

For transplantation, mammary glands containing PN1a tissue were removed from donor mice at 8 weeks post-transplantation, minced into 1 mm fractions with a scalpel and re-transplanted into the cleared fat pads of 3 week-old female Balb/c mice as previously described (18 (link)). At 8 or 16 weeks post-transplantation, inguinal mammary glands containing PN1a outgrowths were fixed in cold 4% paraformaldehyde for 2 h and stained with carmine alum overnight (six mammary glands per timepoint). The next day, glands were dehydrated and imaged on a Leica M165 FC stereoscope (Leica Biosystems) as previously described (19 (link)). After imaging, mammary glands were embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) as previously described (20 (link)). H&E images were captured using an upright Nikon Eclipse microscope (Nikon Instruments). For tumors, mice were palpated twice weekly until tumors were measurable, and then measured three times a week. When tumors reached 1.2 cm in size, mice were euthanized and excised tumors were fixed with 4% PFA overnight and embedded in paraffin for subsequent immunostaining.