Anti rat alexa fluor 647

Cells were seeded on coverslips and were transduced with recombinant lentiviruses. After 16 days, cells were washed with PBS and fixed in 4% formaldehyde for 20 min at room temperature. The cells were then washed three times in PBS and permeabilized in PBS containing 0.2%TritonX-100 and 1% bovine serum albumin (BSA) at room temperature for 30 min. Cells were probed with primary rat antibody to LANA (LN53, Santa Cruz) at 40 °C, and a conjugated secondary anti-rat Alexa Fluor 647 (Jackson Labs) was then applied for detection. To stain the nuclei, the cells were incubated for 30 min with 0.05 µg/ml Hoechst dye (Sigma). Cells were examined and photographed under a confocal laser-scanning microscope (Leica STED Live Imaging).

For the sorting of co-cultured HaCa4 and LEC, and PDV and LECs, cells were stained as previously described [96 (link)] using the primary antibody CD31 (Rat MEC 13.3,1:100, B.D. Franklin Lakes, NJ, USA) and the secondary antibody anti-rat AlexaFluor 647 (1:200, Jackson ImmunoResearch, West Grove, PA, USA). HaCa4 or PDV cells were stained with CFSE Cell Proliferation Kit (ThermoFisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions, and CD31⁺ LECs were sorted with an Aria II FACS sorter (B.D. Franklin Lakes, NJ, USA) excluding dead cells and doublets.