Freshly dissected tibiae were collected from wild type, mutant mice and their control littermates. Bone were processed and imaged as reported earlier8 (link)9 (link). The following primary antibodies were used: Emcn (sc-65495, Santa Cruz, diluted 1:100), Pecam1 conjugated to Alexa Fluor488 (FAB3628G, R&D Systems, 1:100), Pecam1 (553370, BD Pharmingen, 1:100), Osterix (sc-22536-R, Santa Cruz, 1:200), alphaSMA-Cy3 (C6198, Sigma, 1:100), Calcitonin receptor (ab11042, Abcam, 1:75), Dll4 (AF1389, R&D Systems, 1:100), Biotin-conjugated CD45 (553077, Becton Dickinson, 1:100) and Ter-119 (MAB1125, R&D Systems, 1:100). After sections had been washed with PBS for three times, the following secondary antibodies were used: Anti-rat Alexa Fluor 594 (Invitrogen; A21209, 1:400), anti-rabbit Alexa Fluor 546 (Invitrogen; A11035, 1:400), anti-goat Alexa Fluor 488 (Invitrogen; A11055, 1:400) and streptavidin Alexa Fluor 488 (Invitrogen; S11223, 1:400). Immunofluorescent stainings were analysed at high resolution with a Zeiss laser scanning confocal microscope, LSM780.
Proliferating cells were labelled by injecting mice intraperitoneally with EdU (Invitrogen) 3 h before euthanasia. Mice were killed; bones were collected and processed following the protocol mentioned before. For immunostaining EdU using Click-iT chemistry, manufacturer's protocols were followed.
Proliferating cells were labelled by injecting mice intraperitoneally with EdU (Invitrogen) 3 h before euthanasia. Mice were killed; bones were collected and processed following the protocol mentioned before. For immunostaining EdU using Click-iT chemistry, manufacturer's protocols were followed.