Ab41951

Western blotting was performed using the methods reported by Legartová et al. (2014) and Franek et al. (2016) [43 (link),6 (link)]. We used the following primary antibodies: anti-53BP1 (#ab21083, Abcam), anti-α-tubulin (#ab80779, Abcam), anti-MDC1 (#ab11169, Abcam), anti-MDC1 (#ab41951, Abcam), anti-phosphorylated histone H2AX (γH2AX; phospho S139; #ab2893, Abcam), anti-HP1β (#MAB3448, Merck), anti-H3 (#ab7091, Abcam), anti-H3K9me1 (#ab9045, Abcam), anti-H3K9me2 (#ab1220, Abcam), anti-H3K9me3 (#ab8898, Abcam), anti-histone H3K9ac (#06-942, Merck), anti-histone H4K20ac (#701778, ThermoFisher Scientific), anti-histone H4K20me2 (#A-4047-050, Epigentek, Lab Mark a.s.), anti-histone H4K20me3 (#A-4048-050, Epigentek, Lab Mark a.s.). As secondary antibodies, we used anti-rabbit IgG (#A-4914, Merck, Germany; dilution 1:2000), anti-mouse IgG (#A-9044, Merck; dilution 1:2000) and anti-mouse IgG1 (#sc-2060, Santa Cruz Biotechnology, Dallas, Texas, USA; dilution 1:1000).

Neutral red staining, alcian blue cartilage staining, and TUNEL assay, were performed as described previously^{58 (link)}.
Immunolabeling was performed in limb tissue samples fixed in 4% PFA. We employed dissociated cells obtained from squashed interdigital tissue fragments, or vibratome sections permeabilized with Triton X-100 in PBS. The following primary antibodies were employed (dilution 1:100): rabbit polyclonal anti-DNMT1 (NB100–26455; NovusBio.Co. USA) rabbit polyclonal anti-DNMT3A (ab2850; and ab188470; Abcam); rabbit polyclonal anti-DNMT3B (ab2851; Abcam); mouse monoclonal against 5mC (33D3 Eurogentec); rabbit polyclonal anti-MDC1(ab41951; Abcam); mouse monoclonal anti-γH2AX (JBW301, Milipore-Upstate); and rabbit polyclonal anti-Sox9 (Merk-Millipore). Observation were made with a LSM51O laser confocal microscope (Zeiss).
For quantification experiments of 5mC foci or degenerating cells (γH2AX and TUNEL), at least 100 cells were counted in each individual experiment.