Expression 11000xl graphics arts scanner

The fourth mammary fat pads were removed from Lpin1^+/+ and Lpin1^−/− mice and quickly spread onto ice-cold glass slides, and then fixed in Carnoy’s fixative (ethanol:chloroform:glacial acetic acid = 6:3:1, v/v/v) for 2–4 h. The glands were then washed with 70% ethanol for 15 min, rinsed in distilled water once for 5 min, and stained with Carmine Alum (Stem Cell Technologies, Cat. 07070) according to the manufacturer’s instructions. The slides were washed with ethanol (70%, 95% and 100%), cleared with xylene and mounted with neutral balsam. Images were taken an Epson Expression 11000XL Graphics Arts scanner.

Cell counting kit-8 (CCK-8) (MCE, Cat. HY-K0301) and cell proliferation ELISA, BrdU (colorimetric) (Roche, Cat. 11647229001) were used to determine cell proliferation according to manufacturer’s instructions. For anchorage-independent growth in soft agar, the bottom layer was generated by covering 6-well plate with 1.5 ml of 0.5% Nobel Agar (BD, Cat. 214220) in a growth medium that was then solidified at 37 °C for 30 min. 5000 cells were plated on the bottom layers in 1.5 ml of 0.3% Nobel Agar in the growth medium. A layer of growth medium was maintained over the upper layer of agar to prevent desiccation. Cells were maintained at 37 °C for 3–4 weeks for colony formation, followed by staining with nitroblue tetrazolium chloride (1 mg/ml) (Sangon Biotech, Cat. A100329-0500). Images were taken by an Epson Expression 11000XL Graphics Arts scanner.