Slow fibers are specified embryonically and then express ephrin-A3 after birth. (A) Slow myofibers (MyHC-I, red) in the soleus are present at E18.5, but ephrin-A3 staining (green) remains diffuse until P1. (B) Slow myofibers (MyHC-I, red) in the EDL (outline) possess more specific ephrin-A3 (green) expression than the soleus at E18.5, but slow myofibers in the TA have only faint ephrin-A3 expression even at P1. Bars, 100 µm. (C) Transverse cryosections of distal hindlimbs of wild-type pups at E18.5 and P5 stained for ephrin-A3 (green) and MyHC-I (red) to highlight muscle-specific difference in coexpression pattern and intensity. The soleus (S, arrowhead), TA (T, closed arrow), and EDL (E, open arrow) muscles can be identified by the spatial pattern of MyHC-I^+ve myofibers in each section. Bar, 500 µm.