Figure 4

Immunostained sections of axolotl hindlimbs. Longitudinal sections of control (a, d, g) versus 1 day post amputation (dpa) (b, e, h) and 7 dpa (c, f, i) axolotl hindlimbs stained with primary antibodies to nitric oxide synthase 1 (NOS1) (a-c), fibronectin 1 (FN1) (d-f), α-actinin (ACTN) (g-i). Conjugated secondary antibodies were alexa-568 for fibronectin and NOS1, and alexa-488 for α-actinin. Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI). As expected from the proteomic data, fluorescence intensity of NOS1 showed a significant increase compared to control at 1 dpa, then decreased to a level slightly above control at 7 dpa. Fibronectin staining (red) at 1 and 7 dpa showed significant increases compared to controls, while α-actinin staining intensity (green) showed significant decreases.