Fig. 2. Meis expression and regulation by RA during limb regeneration. (A) Comparative results of quantitative RT-PCR amplification of Meis1, Meis2 and Hoxa13 from proximal (P), distal (D) and RA-treated distal (D+RA) medium bud blastema cDNA. (B) Western blot analysis of proximal (P), distal (D) and RA-treated distal (D+RA) blastemas. LC, loading control. RA treatment in A and B was maintained for 3 days before analysis. (C-H) Immunohistochemical detection of Meis-a (red) and nuclei (green) are shown on cryosections of distal blastemas at 4 days post-amputation (C,F), or distal blastemas 3 (D,G) and 6 (E,H) days after RA injection. (F-H) Higher magnification of boxed regions in C-E. (I-K) Hoxa13 in situ hybridization of blastemas treated as in C-E. Bracket in K indicates the distal blastema domain in which Hoxa13 expression is reactivated. (L-N) Anti-Pbx1/2/3/4 immunohistochemistry on proximal (L), distal (M) and RA-treated distal (N) medium bud blastemas, revealing nuclear Pbx localization throughout the blastema.