Figure 5

Validation of endogenous Hoxa3 expression boundary in adult carotid artery of FVB/NTac mouse. (A) Diagram of right carotid segment indicating reporter gene expression domain (blue shading) with anterior boundary at level of lingual artery (la) as observed in Hoxa3-lacZ transgenic mouse (see Fig. 2E for comparison); yellow arrow points to carotid bifurcation into internal (ica) and external carotid arteries (eca), respectively; sections corresponding to levels L1 and L2 as indicated in the cartoon were used for detection of Hoxa3 and Acta2 by immunofluorescence as shown in the three upper (B,B'B") and lower (C,C',C") panels to the right. Both L1 and L2 sections were incubated with Hoxa3-specific and Cy3-conjugated Acta2-specific antibodies; using Cy5-conjugated secondary antibodies, red-fluoresent-labeled Hoxa3 protein was detected only in section L2 (panel C'), whereas green-fluorescent-labeled Acta2 expressed in VSMCs was detected in both L1 and L2 sections (panels B" and C"); accordingly, multichannel composite micrographs (panels B and C) for detecting red (Cy5), green (Cy3) and blue (Hoechst 33342 for nuclear labeling; this is not shown individually) indicate co-localization of Hoxa3 and Acta2 (yellow) in VSMCs only in section L2 (panel C). fa: facial artery; oa: occipital artery.