Pe conjugated anti cd90

For immunostaining, 5-μm-thick keloid tissue sections were fixed and permeabilized with acetone, washed with PBS, and then blocked with 10% normal goat serum for 30 min. To analyze the populations of T helper cells and STAT, the tissues were stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD4 (BD Biosciences, Sparks, MD, USA), phycoerythrin (PE)-conjugated anti-IL-17 (BioLegend, San Diego, CA, USA), or/and PE-conjugated anti-phosphorylated STAT3 tyrosine 705 and PE-conjugated anti-phosphorylated STAT3 tyrosine 727 (BD Biosciences) antibodies. For fibroblast analysis, the tissues were stained with PE-conjugated anti-CD90 (eBioscience) and FITC-conjugated anti-SDF-1 (R&D Systems, Minneapolis, MN, USA) antibodies overnight at 4 °C. The primary antibody was detected using a FITC-conjugated anti-rabbit IgG secondary antibody for 2 h at room temperature, and the nuclei were stained with 4′,6-diamidino-2-phenylindole. The stained tissues were analyzed using a Zeiss microscope (LSM 510 Meta; Carl Zeiss, Oberkochen, Germany) at × 400 magnification.

To analyze the expression of cell surface markers, hBM-MSC or FB were typsinized, re-suspended in PBS containing 2% FBS, and stained with the following antibodies for 30 min at 4 °C: PE-conjugated anti-CD228 (R&D Systems, Minneapolis, MN, USA), APC-conjugated anti-CD106 (BioLegend, San Diego, CA, USA), PE-conjugated anti-CD90, PE-conjugated anti-CD73, APC-conjugated anti-CD105, PE-conjugated anti-CD200, PE-conjugated mouse IgG1 isotype control, or APC-conjugated mouse IgG1 isotype control (all from eBioscience, San Diego, CA, USA). Flow cytometry was performed on FACSCalibur^TM (BD Biosciences, Sparks, MD, USA) and analyzed with CellQuest Pro^TM software (BD Biosciences).