Brdu solution

To quantify the proliferation of NSCs, a bromodeoxyuridine (BrdU) assay was used. After incubating NSCs in OEC CM or OEC Fractioned CM for 3 or 7 days, media from cells was removed and discarded. Cells were incubated in 100μM BrdU solution (Invitrogen) for 1.5 hours at 37°C. Cells were subsequently fixed in 10% Formalin then washed with 0.1M PBS and 1% Triton X-100 solution. Cells were incubated in 1N HCl for 10 minutes on ice, then with 2N HCl for 10 minutes at room temperature and 20 minutes at 37°C. HCl was removed and borate buffer (0.1M) added for 12 minutes at room temperature. Cells were washed and blocked with blocking buffer (0.1M PBS, 1% Triton X-100, 1M glycine, 5% NGS) for 1 hour at room temperature and incubated with anti-BrdU antibody (rabbit α BrdU, 1:1000) overnight. Antibody was detected with Alexafluor 647 antibody and visualized using Zeiss Axiovert microscope using Cy5 channel. The area of BrdU positive cells was measured per 20× frame and normalized by the area of GFP positive cells. Area fluorescence measurements were made using calibrated measurement software tool in Axiovision program. Area was used rather than number of cells as it was difficult to count cells in the neurosphere.

Pregnant dams (E13.5) were injected once with BrdU solution (50 µg/g body weight, Invitrogen 00-0103), harvested one hour later and fixed overnight in 4% PFA. Heads were infiltrated with sucrose/gelatin and 6 µm cryosections prepared as described above. DNA was denatured by incubating the slides in 1N HCl at 48° for 30 minutes, followed by neutralization in PBS. BrdU was detected as described above using a mouse monoclonal antibody, MoBu-1 (Invitrogen B35128) at 1:100 and Alexa Fluor® 594 goat anti-mouse (Invitrogen A11032). For analysis at E11.5, cryosections were prepared and phospho-Histone H3 was detected as described above. To differentiate between sensory and non-sensory duct domains, we co-stained the sections to detect SOX2 also as described above. E11.5 data came from 8 sections in each of 6 controls and 6 Fgf10 null mutants. E13.5 data came from 5 sections in each of 4 controls and 4 Fgf10 null mutants. Student’s t-test (unpaired, two-tailed; Prism 6.0) was used to compare the mean number of BrdU or phospho-Histone H3-positive cells per unit area in control and Fgf10^−/− samples. SOX2-positive and SOX2-negative domains were considered separately.

OAF cells from IFT80^fl/fl mice were infected with Ad-Cre (with or without SAG treatment) or Ad-GFP and cultured for 2–3 days. The cultures were incubated with BrdU solution (1:100, 000103, Invitrogen, USA) for 20 h, and stained with a BrdU staining kit (1:100, MA3071, Invitrogen, USA) according to the manufacturer’s instructions. BrdU positive and total cell numbers were counted in 10 images per subject. The number of BrdU-positive cells was indicated as a percentage of the total cell number. BrdU assay was repeated on three independent samples for each experimental group.