Bu 33

The β-galactosidase activity assay^{20 (link)} was performed at pH 6 in vibratome sections of limb autopods fixed in 4% glutaraldehyde.
Neutral red staining, TUNEL assay, and electron microscopy were performed as described previously^{2 (link)}.
Immunolabeling was performed in limb tissue samples fixed in 4% PFA. We employed both squashed interdigital tissue fragments or vibratome sections permeabilized with Triton X-100 in PBS. The following antibodies were employed: rabbit monoclonal anti-UHRF1 (DSG8E, Cell Signaling), rabbit polyclonal anti-UHRF2 (TA337863, OriGene); mouse monoclonal anti-UHRF2 (sc-398953, Santa Cruz Biotechnology); mouse monoclonal anti-5-methylcytosine (5-mC; 33D3, Eurogentech); rabbit polyclonal anti-SOX9 (AB5535,Milipore); mouse monoclonal anti-γH2AX (JBW301, Milipore-Upstate); and mouse monoclonal anti-BdrU (BU-33, Sigma Aldrich). Counterstaining to distinguish nucleus and cytoplasm was performed using fluorescent-phalloidin (Sigma Aldrich) or DAPI (Vector Laboratories). Observation were made with a LSM51O laser confocal microscope (Zeiss).

The following monoclonal anti-bromodeoxyuridine antibody clones were tested in this study: BMC9318 (Roche), Bu-33 (Sigma Aldrich), B44 (Becton Dickinson), Bu6-4 (Genetex), Bu20a (BioLegend) and Bu5.1 (Millipore). As a secondary antibody, we used anti-mouse antibody conjugated with the fluorochrome Alexa Fluor 488 (1:100, Jackson ImmunoResearch).

Immunodetection of nascent rRNA was performed by incorporation of 5-fluorouridine (5-FU), according to the method described [54 (link)]. Briefly, cells growing on coverslips were incubated with 2 mM 5-FU (Sigma) for 15 min, then washed with cold PBS and fixed in 4% paraformaldehyde and 1% Triton X-100 in PBS for 10 min. Subsequently, the cells were immunofluorescently stained with a specific monoclonal antibody for halogenated uridine (1:400, Sigma [BU-33]). Mounting and nuclei counterstaining and immunofluorescent image were performed as describe as above. Quantification of incorporation of 5-FU into rRNA was using Olympus cellSens^™ Dimension software (Olympus Life Science).