Anti f4 80 brilliant violet 421

Cells were stained with fluorochrome-labeled mAbs and analyzed with an LSRII flow cytometer (BD). Flow cytometric analysis was performed using FlowJo software. The following fluorochrome-labeled mAbs were purchased from Biolegend, Inc. and used according to the manufacturers’ protocols: anti-CD11b-FITC (101205, RRID: AB_312788), anti-CD206-APC (141707, RRID: AB_10896057), anti-Ly6C-APC-Cy7 (128026, RRID: AB_10640120), anti-Ly6G-PE (127608, RRID: AB_1186099), anti-F4/80-Brilliant Violet 421 (123132, RRID: AB_11203717), anti-CD64-PE (139303, RRID: AB_10613467), and anti-Siglec-F-APC (155507, RRID: AB_2750236).

The immunization procedures were similar to that described in the previous section. However, the testing area was protected by paper and then covered with an occlusive dressing and the cage was covered to protect from light. After euthanasia, a section of the immunization site was rinsed with PBS to remove residual formulation and then harvested. The skin was macerated and incubated at 37°C for 1 hr in collagenase type IA (C9891, Sigma-Aldrich, St. Louis, MO) digestion buffer. A single cell suspension was fixed in 8% paraformaldehyde at room temperature. Cells were then permeabilized using saponin (S-7900, Sigma-Aldrich, St. Louis, Mo) buffer and incubated with anti-CD16/CD32 before being stained with anti-CD207-Alexa Flour 488 (eBioscience, San Diego, CA) for 30 minutes followed by washing and staining with anti-CD11c-APC, anti-CD11b-PE-Cy7 (BD PharMingen, San Diego, CA) and anti-F4/80- Brilliant Violet 421 (Biolegend, San Diego, CA). Cells were then analysed using a FACSCanto™ II flow cytometer (BD Biosciences, San Jose, CA). Data was analysed using FlowJo 7.6 analysis software (Tree Star, Inc., Oregon, USA).