Although much physiology in resistance vessels has been attributed to cytoplasmic connection between endothelial cells (EC) and vascular smooth muscle cells (VSMC), little is known of protein expression between the two cell types. In an attempt to identify proteins between EC and VSMC, mouse cremaster arterioles were stained with phalloidin-Alexa594 and viewed on a confocal microscope which resolved "actin bridges" within the internal elastic lamina between EC and VSMC. To determine the incidence of protein, the pixel intensity from the antibodies on actin bridges were compared to the pixel intensity from antibodies within EC or VSMC. N-cadherin, desmin, connexin (Cx)40 and Cx43 and phosphorylated Cx43 at serine 368 were identified on actin bridges but NG2, CD31 and Cx45 were not evident. Connexin37 expression was more variable than the other connexins examined. Using this method on rat mesentery, we confirm the previously published predominance of Cx37 and Cx40 at the myoendothelial junction that was determined using electron microscopy. We conclude that this new method represents an important screening mechanism in which to rapidly test for protein expression between EC and VSMC, and possibly a first-step in quantifying protein expression at the myoendothelial junction.