JCS037481 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Immunohistochemistry of freshly isolated cells after cytospin. CD-31 isolated cells (EC) or NG2-isolated cells (VSMC) were cytospinned onto plus coated slides and immunocytochemistry performed as described in the Materials and Methods. eNOS was used to identify EC and α-actin was used to identify VSMC. The total number of cells, identified by both nuclei staining and attached beads, were counted and the cells with fluorescence from eNOS or α-actin were noted. Green is SYTOX-nuclear stain; red is the antibody of interest.

• Supplemental Figure S2 -

  Fig. S2. Cremasteric microvascular cell isolation. (A) NG2, when viewed along the length of the whole-mount vessel (green) or in (B) transverse frozen sections (red is NG2, green is IEL). (C) Beads coated with NG2-pulled-down cells with a horse-shoe-type appearance. (D) When the NG2-isolated cells (right) were run on an immunoblot with isolated CD31-isolated cells (left), the NG2-isolated cells stained for NG2 (top) and desmin (bottom). (E) CD31 staining is evident along the length of the vessel (green) as well as in (F) transverse frozen sections (red is CD31, green is IEL). (G) CD31-coated magnetic beads pulled-down cells that appeared more rounded than those isolated by NG2 beads. (H) The CD31-isolated cells were run on an immunoblot (left) with NG2-isolated cells (right). Only the CD31-isolated cells stained for CD31 (top) and VE-cadherin (bottom). Bars in A-C are 10 µm whereas bar in A is representative for E and bar in B is representative for F; bar in G is 15 µm.

• Supplemental Figure S3 -

  Fig. S3. Griffonia lectin demonstrates that isolated cremasteric endothelial cells are microvascular phenotypes. Cultured EC originating from angiogenic explants of aorta (A,B) or from magnetic bead isolated cremasteric EC (C,D) were probed with Griffonia simplicifolia conjugated with Alexa Fluor 594 (A,C) or Helix pomatia conjugated with Alexa Fluor 594 (B,D). Although staining for both lectins is observed in EC from angiogenic explants (A,B), only staining for Griffonia is observed in the isolated cremasteric EC (C,D). Scale bar in A is 30 µm and is representative for all images.

• Supplemental Figure S4 -

  Fig. S4. Restricted dye transfer in cremasteric vascular-cell co-culture. EC were loaded via pinocytotic methods with both biocytin and Cy3. After loading of dye into the EC, the entire VCCC was fixed with 2% paraformaldehyde and probed with streptavidin Alexa Fluor 488 so as to expose the location of the biocytin. Similar to previous results using angiogenic explants of aortic EC and VSMC, biocytin (green) was present in both EC and VSMC regardless of the connexin composition of the gap junction at the in vitro MEJ; however, Cy3 (red) dye movement to VSMC was only apparent when Cx43 homotypic gap junctions were present at the in vitro MEJ (i.e. EC Cx40^−/−).