Fig. 1. Phenotypic verification and connexin expression in cremasteric vascular-cell co-culture. En face staining for acetylated low-density lipoprotein (LDL) demonstrated ECs on the top of the Transwell insert, with no observed staining for -actin (A). (A) Transverse sections of the VCCC (top is ECs, bottom is VSMCs) double stained with phalloidin (F-actin, green) and VE-cadherin (red) demonstrated distinct EC localization in the pores of the Transwell. In the adjacent immunoblot, CD31 stained only the EC isolates. (B) When examining VSMCs en face, acetylated LDL was not detected; however, -actin was found throughout the monolayers. Transverse sections of VCCC revealed VSMC extensions, which stained with desmin (red), following the F-actin (green) within the pores of the Transwell. The immunoblot, which was probed for desmin, demonstrates that only VSMC isolates express this phenotypic marker. (C-F) In transverse sections of the cremasteric VCCCs, Cx37 (C), Cx40 (D), Cx43 (E) and Cx45 (F) were observed. Arrows demonstrate connexin expression within the pores of the Transwell. (G) Single pores of the Transwell were double stained for both Cx40 (red) and Cx43 (green) to demonstrate a lack of re-arrangement of connexins at the in vitro MEJ when Cx43 or Cx40 was eliminated. Scale bars: 75 µm (en face view in A,B); 10 µm (transverse view from A,B; C-F); 2.5 µm (G).