Bromodeoxyuridine (brdu)

Bromodeoxyuridine (BrdU) was used to identify proliferating cells in the first 7 days post-SCI. Intraperitoneal injections of 50 mg/kg BrdU (Roche, Basel, Switzerland) were pulsed every day for 7 days, allowing 7 additional days for unbound BrdU to wash out before exogenous E14 spinal progenitors were implanted, as done by others.^{60 (link)} In rodent injury models, endogenous neural progenitor populations peak within the first 7 days after injury and gradually decrease with time.^{61 (link)} As such, a conservative estimate of 7 days post-SCI for daily BrdU injections was chosen for labeling proliferating cells. Any cells that co-localized with BrdU and neuronal lineage markers described below were considered to arise from endogenous spinal progenitors giving rise to new cells along the neuronal lineage. Tissue sections requiring BrdU identification were first denatured in 2N HCl for 1 hour at 37 °C followed by neutralization in 2 five minute rinses of 0.1M borate buffer to facilitate antigen retrieval. Samples were incubated for 1 hour at room temperature in rat anti-BrdU (1:200, Abcam, Cambridge, UK) antibody followed by appropriate fluorophore-conjugated goat anti-rat secondary antibody before proceeding with additional immunohistochemistry of specific cell phenotypes described below.

The S-phase labelling index and S-phase length were obtained by a dual S-phase marker labelling technique as described by Gonsalvez et al. (2013 (link); 2014 ). Briefly, pregnant dams between 10.5–16.5 days post-fertilization were injected intraperitoneally with the first S-phase marker, bromodeoxyuridine (BrdU; Roche Diagnostics), at 100 μg/g body weight followed, 2 hours later, by the second S-phase marker, ethyldeoxyuridine (EdU; Invitrogen) at 50 μg/g body weight. After a further 30 minutes, the dams were killed and embryos were collected for immunohistochemistry analysis.