Skeletal whole-mount staining

For skeletal preparation, whole-mount skeletal preparations of 5–7-day-old Piezo1^fl/fl and Piezo1^Ocn/Ocn mice were prepared by removing the skin and internal organs of the mice before immersion in 95% ethanol for 1–3 days. Specimens were stained with 0.015% Alcian Blue (Sigma) in 80% ethanol with 20% acetic acid. After staining, specimens were washed twice in 95% alcohol for 2 hr, cleared in 1% KOH for 5 hr and stained in 0.005% Alizarin red (Sigma) in 1% KOH for 1 hr. They were then cleared through 20%, 50%, and 80% glycerine in 1% KOH, then stored in 100% glycerine.