Lymphangiogenesis Assays Using Primary Mouse LEC Figure 2. Purity of isolated embryonic dermal LEC and BEC populations assessed by immunostaining. Analysis of markers indicative of LEC and BEC identity on cells isolated from E16.5 dermis and cultured in EGM-2MV for 72 h. LEC are uniformly Prox1 and CD31 positive and depleted of hematopoietic cells. BEC are uniformly CD31, CD34, VEGFR2 and Nrp1 positive and devoid of LEC and hematopoietic cells. Scale bars represent 40 mm. doi:10.1371/journal.pone.0040497.g002 Reagents and Antibodies We used Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma), Hank’s Balanced Salt Solution (HBBS, Sigma), recombi- nant human FGF basic 146 aa (R&D Systems), recombinant human VEGF-C (R&D Systems), SU5402 (Merck), SU5416 (Sigma-Aldrich), MAZ51 (Sigma-Aldrich), rabbit anti-mouse Prox1 (AngioBio), goat anti-human Prox1 (R&D Systems), rabbit anti-mouse LYVE-1 (AngioBio), rat anti-mouse CD31 (BioLe- gend), rat anti-mouse CD45 (BD Pharmingen), rat anti-mouse CD34 (eBioscience), rat anti-mouse F4/80 (Invitrogen), goat anti- PLoS ONE | www.plosone.org 3 July 2012 | Volume 7 | Issue 7 | e40497