Figure 4

Detection of connexin isoforms in RTEC by RT-PCR and immunocytochemistry. RT-PCR (A, B) or immunocytochemistry (C-H) were used to identify connexin isoform expression changes between RTEC grown on collagen or LM-332/collagen. Total RNA was subjected to reverse transcription followed by PCR for Cx26, Cx43, Cx46 or β-actin (A, B). No differences in mRNA products from RTEC grown on either matrix were observed. Representative immunocytochemical micrographs of RTEC grown on collagen (C, E, G) or LM-332/collagen matrices (D, F, H) stained with antibodies against Cx26, Cx43, or Cx46 are shown. On the collagen matrices, all connexin isoforms display a perinuclear staining pattern, with a pericellular staining pattern also evident in the Cx46 micrograph. On the LM-332/collagen matrices, a noticeable shift in pericellular staining is evident in Cx26 and Cx43 micrographs, whereas the most evident staining of Cx46 is perinuclear. Growth of RTEC in the presence of LM-332 alters the spatial pattern of connexin isoform expression. Arrowheads denote pericellular staining and arrows denote perinuclear staining. Bar in C represents 20 μm and is relevant to C – H.