Immunofluorescence Staining in Planarians

Immunofluorescence staining was performed as described before⁴¹ . Different treatments were used for different antibodies. Following antibodies were used: Phospho-histone H3 (Ser10) (H3p) (1:1000, Abcam, ab32107). Planarians were sacrificed in 5% N-acetyl cysteine (NAC) for 5 min at room temperature. After being bleached with 6% H₂O₂ overnight, animals were washed with PBSTx (0.3% Triton X-100). After 2 h of blocking with 1% BSA, animals were incubated with primary antibody overnight, then washed with PBSTx more than six times and 1 h each. Blocking with 1% BSA for 1 h was performed before the incubation of the fluorescent secondary antibody cy3-labeled goat anti-rabbit IgG (1:1000, Beyotime, A0516) overnight. After extensive wash with PBSTx for more than 6 h, the animals were mounted with 80% glycerol containing Hoechst 33342 (10 µg/ml, Invitrogen, H3570). Quantification of fluorescent signals was analyzed by ImageJ software (V1.5) as previously described^{43 (link)}.