Rat anti cd31 apc

For FACS analysis of tumors and control tissues (back skin and abdominal skin, respectively), the tissue was dissected, minced, and digested in a collagenase solution [5 mg/ml Collagenase II (Sigma), 40 µg/ml DNaseI (Roche)] for 30 min at 37°C. The digested tissue was passed through a cell strainer before erythrocyte lysis with PharmLyse (BD). After washing and a second filtration step, the cell suspension was labeled with fluorescently tagged antibodies [hamster anti-podoplanin-PE (1:400, clone 8.1.1, eBioscience), rat anti-CD31-APC (1:300, clone MEC13.3, BD), rat anti-CD45-APC/Cy7 (1:200, clone 30-F11, Biolegend), and rat anti-PDL1-PE/Cy7 (1:200, clone 10F.9G2, Biolegend)]. 7AAD (Biolegend) was used for life/dead discrimination. Inguinal LNs were processed essentially as described before (31 (link)). In brief, the capsule of dissected LNs was ruptured and the tissue was digested with 1 mg/ml Collagenase IV (Gibco)/40 µg/ml DNaseI for 20 min at 37°C to release the majority of the immune cells. The remaining stromal fragments were washed twice, digested with 3.5 mg/ml Collagenase IV/40 µg/ml DNaseI for 15 min at 37°C, and disaggregated by pipetting in presence of 0.5 mM EDTA. After filtration, the stromal cell enriched cell suspension was stained as described above. Data were acquired on a FACS CANTO (BD) and analyzed using FlowJo (Treestar Inc.).

Ears were digested with collagenase IV (Invitrogen) as described [19 (link)]. Ear single cell suspensions were stained with the following antibodies: rat anti-CD45-APC-Cy7 (1:250, Biolegend), rat anti-CD31-APC (1:100, BD), hamster anti-podoplanin (1:100, clone 8.1.1, Developmental Studies Hybridoma Bank, University of Iowa) and anti-hamster-Alexa 488 (1:200, Invitrogen). FACS analysis was performed on a BD Fortessa analyzer (BD Biosciences) using the FACSDiva software. Data were analyzed with FlowJo software (TreeStar).