C3h hej c3h mice

All procedures were reviewed and approved by the Institutional Animal Care and Use Committee at Stony Brook University (IACUC). The experimental design of this study has been described, in part, in a previous study which also contains a subset of the μCT data presented here [19 (link)]. Thirty-four female, 7wk old C3H/HeJ (C3H) mice, purchased from The Jackson Laboratory (Bar Harbor, ME), were weight-matched and assigned to either baseline controls (BC, n = 10), age matched controls (AC, n = 12), or hindlimb unloaded (HLU, n = 12) groups. All mice were weighed prior to group assignment. The study commenced when mice were 9wk old. The duration of the experimental protocols was 3wk. All mice were individually housed and had access to standard rodent chow and water ad libitum. Weight-bearing was removed through hindlimb unloading (HLU), a ground-based model stimulating many physiological effects of microgravity [20 (link)–22 (link)]. Following euthanasia, femora were fixed in 10% formalin for 24h and stored in 70% ethanol for ex-vivo micro-computed tomography (μCT), histomorphometry, and histology. Immediately after harvesting, bone marrow was flushed with Dulbecco's Modified Eagle Medium (DMEM, Gibco, Carlsbad) from both tibiae.

The breeding pair of CBS+/− and C3H/HeJ (C3H) mice was obtained from Jackson Laboratories. After 4 weeks, mice were weaned and genotyped. For genotyping by PCR using specific CBS primers, samples of mice tails were collected. The PCR products were run on 1.2% Agarose gel (prepared in TAE buffer, pH 8.4) with ethidium bromide. The images were recorded in gel documentation system (Bio‐Rad, Hercules, CA) and the genotyping data were confirmed as per Jackson Laboratories Protocol.