Ab24607

Cells were lysed in TNT lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 50 mM NaF, 1 mM Na₃VO₄, 1 mM PMSF, and protease inhibitor cocktail (Sigma)) and protein amounts from cell lysates were measured using the DC protein assay (Bio-Rad). Equivalent amounts of proteins were subjected to SDS-PAGE, transferred to Immunobilon-P membranes (Millipore), immunoblotted with anti-GFP antibody (JL-8, 632381, Takara, 1:1000), anti-Cre recombinase antibody (ab24607, Abcam, 1:1000), anti-Hsp90 antibody (675402, Biolegend, 1:1000), and developed with ECL (Thermo fisher scientific). Immunoblotting with anti-HSP90 antibody was used as a loading control. Alternatively, dissected femurs and soft tissues were homogenized in RIPA lysis buffer (89900, Thermo fisher scientific) and tissue lysates were subjected to immunoblotting analysis.

Fish samples were fixed in formalin solution (Sigma-Aldrich) and sectioned at 5-µm thickness using a microtome, followed by hematoxylin and eosin (H&E) (Sigma-Aldrich) and immunofluorescent staining. Immunofluorescent staining was carried out as previously described (Yan et al., 2015 (link)). The primary antibodies used included rabbit anti-PCNA at a dilution of 1:200 (Santa Cruz Biotechnology, SC-7907), rabbit anti-Caspase 3 at a dilution of 1:200 (BD Biosciences, 559565), mouse anti-GFP at a dilution of 1:500 (Millipore, MAB3580), rabbit anti-RFP at a dilution of 1:100 (Abcam, ab62341), mouse anti-DsRed at a dilution of 1:100 (Santa Cruz Biotechnology, sc390909) and mouse anti-Cre antibody at a dilution of 1:200 (Abcam, ab24607).