Mgk s

After washing in PBS, the dissected mammary glands and tumors were fixed in 4% paraformaldehyde–PBS overnight and for 2 days, respectively. After washing in PBS, samples were dehydrated gradually from 70% ethanol to 100% ethanol and then from 50% xylene in ethanol to 100% xylene, following paraffin replacement using a Leica ASP300 fully automatic closed tissue processor and paraffin-embedding using a Leica EG1160. The paraffin-embedded tissues were then cut into 5-μm thick sections using a Leica SM200R sliding microtome. The sections were gradually deparaffinized in xylene and then with ethanol, gradually decreasing from 100% to 50% ethanol, and washed with distilled H₂O, and then stained in hematoxylin solution (0.25% hematoxylin (Nacalai Tesque), 0.05% sodium iodate (Nacalai Tesque), 12.5% potassium alum (Wako), and 0.25% citric acid (Wako)) for 10 min. After washing in distilled H₂O, sections were blued in 0.1% saturated lithium carbonate at 37 °C for 5 min and then washed in distilled H₂O and stained in eosin solution (1% eosin (Wako) and 0.02% glacial acetic acid) for 10 min. After washing in 90% and 100% ethanol, sections were soaked in xylene for 5 min and then mounted in MGK-S (Matsunami).

The dissected mammary glands were spread on a glass slide and fixed using Carnoy’s fixative (60% ethanol, 30% chloroform, and 10% glacial acetic acid) overnight at room temperature. Fixed tissues were washed in 70% ethanol, gradually rehydrated to distilled H₂O, and then incubated in carmine alum solution (0.2 wt% of carmine (Sigma) and 0.5 wt% of aluminum potassium sulfate (Wako) in distilled H₂O) for 2 h to overnight at room temperature. After gradual dehydration from 70% ethanol to 100% ethanol, fat pads were cleared overnight in xylene and mounted in MGK-S (Matsunami, Osaka, Japan).

Muscle tissues were harvested from 4-month-old L-MPZ WT, Het, and Hom mice, and flash-frozen using dry ice–isopentane. Sections (10 µm) were cut by cryostat and placed on FRONTIER slide glass. For histological staining, slides were incubated with Mayer’s hematoxylin solution (Fujifilm Wako Pure Chemical Co.) for 15 min, followed by a 10 min wash. Slides were then incubated in 1% eosin solution (Fujifilm Wako Pure Chemical Co.) for 1 min. Next, slides were washed with tap water for 10 min. Afterwards, they were dehydrated successively with 70, 80, and 90% ethanol (EtOH) for 1 min, three times with 100% EtOH for 1 min, and three times with xylene for 1 min. Samples were mounted with MGK-S (Matsunami Glass) and covered by cover slips, and observed using a BZ-X700 microscope (Keyence).