Pecam 1 mec13.3

Immunostaining was performed in cryostat sections, as described [9 (link),11 (link)]. Mac-1 (M1/70), Ly-6G (1A8), and PECAM-1 (MEC13.3) from BD Biosciences, MMP-2 (Ab19167; Millipore), and MMP-9 (AF909; R&D Systems) antibodies were used at optimal dilutions. Sections were blindly evaluated by counting ten HPFs/section in triplicates. Dual/triple staining was detected by immunofluorescence with Alexa Fluor 594-red anti-rabbit IgG (H+L) and Alexa Fluor 488-green anti-rat IgG (H+L) (Molecular Probes); Vectashield mounting media with DAPI (Vector Laboratories) was used for nuclear staining. Slides were analyzed using a Leica Confocal Microscope.

Human primary dermal lymphatic ECs (HDLEC from juvenile foreskin, cat. no. C12216) were obtained from PromoCell. Cells were seeded on bovine Fibronectin (F1141, Sigma) coated dishes in complete ECGMV2 medium (PromoCell) supplemented with 25 ng ml⁻¹ of VEGF-C (2179-VC, R&D Systems) and used after three to four passages. For assessing proliferation, cells were cultured under confluent contact-inhibited conditions (6.3 × 10⁴ cells per cm²). Murine primary dermal lymphatic ECs were isolated from the tail skin of 6 weeks old Vegfr3^flox/flox;R26-mTmG;Prox1-CreER^T2 female by sequential selection with PECAM1 (Mec13.3, Pharmingen) and LYVE1 (Aly7, Abnova) antibodies bound to Dynabeads, as described previously^{50 (link)}. Mouse LECs were cultured on 0.5% gelatin-coated dishes and passaged 1:3 after 4 days. In vitro gene deletion was induced by adding 1 µM 4-OHT (H7904, Sigma) to the culture medium. Notch signaling was inhibited by 24 h treatment with 10 µM DAPT (CAS 208255-80-5, Calbiochem). All cells were cultured at 37 °C in a humidified atmosphere with 5% CO₂.