Poloxamer 407 (BASF, Ludwigshafen, Germany; 25% w/w) was added to isotonic phosphate-buffered saline (PBS, pH 7.4, 4°C) with gentle mixing until complete dilution [18 (
link)]. Then, rhPTH (1–34) (Lilly, Fegersheim, France) or
IGF-1 (ab198570, Abcam, Cambridge, USA) was added to the prepared
Poloxamer 407 solutions, and the final concentrations of
IGF-1 and rhPTH (1–34) were 60 μg/ml and 80 μg/ml, respectively.
The
Pth1r or
IGF-1r siRNA (Sigma-Aldrich, USA) transfection complex was prepared according to the manufacturer’s instructions. In brief, 20 μg of siRNA (sequences are provided in
Table 1) was dissolved in 20 μl of RNase-free water, followed by the addition of 10 μl of Entranster
TM-in vivo (Engreen, Beijing, China) and gentle mixing; the mixture was then incubated for 15 min at room temperature. Finally, 20 μl of
Poloxamer 407 (BASF, Ludwigshafen, Germany, 50% w/w) was added to the solution with sufficient mixing.
Zhang W., Liu C., Hai B., Du G., Wang H., Leng H., Xu Y, & Song C. (2016). A Convenient In Vivo Model Using Small Interfering RNA Silencing to Rapidly Assess Skeletal Gene Function. PLoS ONE, 11(11), e0167222.