Fitc conjugated anti cd14

The following antibodies were purchased from BD Pharmingen, unless otherwise noted: BV421-conjugated anti-CD3 (#562426); BV421-conjugated anti-CD4 (BioLegend, #300532); BV421-conjugated anti-CD56 (BD Horizon, #562751); BV421-conjugated anti-CD86 (#562432); BV421-conjugated anti-HLA-DR (BD Horizon, #562804); BV421-conjugated IgG1, κ Isotype Control (BioLegend, #400158); BV421-conjugated IgG2a, κ Isotype Control (BD Horizon, #562439); BV421-conjugated IgG2b, κ Isotype Control (BioLegend, #400342); FITC-conjugated anti-CD4 (#555346); FITC-conjugated anti-CD14 (#555397); FITC-conjugated anti-CD15 (#562370); FITC-conjugated anti-CD19 (#555412); FITC-conjugated anti-CD25 (#555431); FITC-conjugated anti-CD69 (#555530); FITC-conjugated IgG1, κ Isotype Control (#555748); FITC-conjugated IgG2a, κ Isotype Control (#555573); PE-conjugated anti-CD8 (#555367); PE-conjugated anti-CD11b (BioLegend, #301406); PE-conjugated anti-Cd11c (#555392); PE-conjugated IgG1, κ Isotype Control (#555749); APC-conjugated anti-CD3 (#555335); APC-conjugated anti-CD11a (R&D Systems, #FAB3595A); APC-conjugated anti-HLA-DR (#559866); APC-conjugated IgG1, κ Isotype Control (#555751); and APC-conjugated IgG2a, κ Isotype Control (BioLegend, #400220).

Freshly excised corneas were washed in PBS and fixed with 4% paraformaldehyde for 20 minutes and permeabilized with 0.5% Triton X-100 for 10 minutes. Whole corneas were then immunostained with FITC-conjugated anti-CD14 (#123308) and PE-conjugated anti-CD11b (#101207) (Biolegend, San Diego, CA, USA) overnight at 4°C to detect myeloid progenitors and mounted onto slides with mounting medium (Vector Laboratories, Burlingame, CA, USA) and visualized using a confocal microscope (Leica TCS-SP5; Buffalo Grove, IL, USA) at ×20 magnification. Corneal sections fixed in 4% paraformaldehyde were stained with hematoxylin and eosin. Images were obtained using a bright field microscope (Nikon Eclipse E800; Melville, NY, USA) at ×20 magnification.