Characterization of circulating precursors cells (A-D).

Morphological (A) and immunohistochemical (B) analysis of cryosections from H. medicinalis body wall. The circulating precursors (arrowhead) visible in lumen of neo-vessels are highly positive for anti-CD45 antibody. Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI; blue). (C-D) Western blot analysis. A protein extracts from the body wall of untreated (n.t) and MWCNTs treated leeches from 1 to 12 hours and from 1 to 5 weeks were probed with the anti-CD45 antibody. The housekeeping protein D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. In all samples, the anti-CD45 detected specific immunoreactive band 24 of about 145 kDa (C), according to the molecular weight ladder (kDa). CD45 protein was quantified by densitometry from three experiments. *P<0.05 compared with untreated leeches (D). Bars in A-B: 10μm.