Fluorescein isothiocyanate (fitc)

Cells were plated directly on a coverslip and allowed to adhere with overnight incubation. The following day, the cells were irradiated and fixed with 4% paraformaldehyde for 10 minutes at room temperature. Cells were then washed with PBS and permeabilized with 70% ethanol overnight at 4°C and for 20 minutes with 0.1% Igepal at room temperature. Cells were washed, blocked with 2% BSA for 1 hour and incubated overnight with 1:1,000 Aurora Kinase A antibody (Cell Signaling Technology). Centrosomes were visualized by 1 hour incubation with a 1:500 AlexaFluor 594 fluorochrome (Invitrogen). Cells were then incubated with 1:500 alpha tubulin (Santa Cruz Biotechnology) for 1 hour at room temperature. Mitotic spindles were visualized by 1 hour incubation with 1:600 FITC (Jackson ImmunoResearch), and DNA was stained with 1 μg/mL DAPI (RRID:AB_2893474; Sigma). Pictures were captured with a Leica microscope.
BRCA1 foci were visualized by incubating 1:500 BRCA1 antibody overnight (Santa Cruz Biotechnology) and 1:600 FITC (Jackson ImmunoResearch) for 45 minutes at room temperature. Foci for at least one hundred cells were manually counted per experiment using ImageJ software. Each foci experiment was repeated at least once.

Standard histology and immunostaining protocols (including H&E and Trichome) were performed. Briefly, immunohistochemical analysis was performed on 5- to 10-μm-thick sections of mouse skin. The following primary antibodies were used: mouse monoclonal anti-Ki-67 (1:100; Novocastra), rabbit polyclonal anti-GFP (1:200; Abcam), rabbit polyclonal anti-Sdf1 (1:100; Abcam), mouse monoclonal anti-α smooth muscle actin (1:100; Abcam), rabbit polyclonal anti-CD31 (1:100; Abcam), rat anti-mouse CD45 (1:100; BioLegend), and rabbit polyclonal anti-Cxcr4 (1:100; Abcam). Immune complexes were detected with secondary antibodies conjugated with either Cy3, Cy5, fluorescein isothiocyanate (Jackson ImmunoResearch), or horseradish peroxidase (Vector Laboratories). In situ hybridization on Cxcl12 was performed per the manufacturer's instructions (Advanced Cell Diagnostics); a positive control probe was directed against RNA polymerase, and a negative control probe was directed against bacterial DapB. After staining, images were directly analyzed on an AxioM1 microscope equipped with a CCD digital camera (Carl Zeiss). The number of Ki-67⁺ cells was counted for every 250 μm of cartilage, measured at the distal tip, for each sample. A minimum of four different samples was used for each time point and then averaged.