Rat anti mouse cd206

The 4T1 murine breast cancer and RAW 264.7 murine macrophage cells (CD206-positive 7 (link)) were purchased from American Type Culture Collection (ATCC, Manassas, VA). Cells were grown in RPMI 1640 medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) at 37°C in a humidified atmosphere containing 5% CO₂.
For overlay staining of CD206 and F4/80, RAW 264.7 cells grown on 35 mm MatTek glass-bottom culture dishes were incubated with rat anti-mouse CD206 (BioLegend, San Diego, CA) and rabbit anti-mouse F4/80 (Abcam, Cambridge, MA) primary antibodies for 1 h at room temperature, and then incubated with dye-conjugated secondary antibodies for 1 h. Cells were mounted with medium containing DAPI (Vector Laboratories, Burlingame, CA) and examined under a confocal microscope (Wetzler, Heidelberg, Germany).
To determine the immunoreactivity of Dye-anti-CD206, RAW 264.7 or 4T1 cells were incubated with Dye-anti-CD206 (10 nmol/L in PBS) or Dye-IgG (10 nmol/L in PBS) for 1 h at room temperature. After washing with PBS and mounting with medium containing DAPI, cells were examined under a confocal microscope.

Ex vivo immunofluorescence staining of 4T1 tumor or normal tissues was performed to validate the results of in vivo studies. The experiments were performed, and the staining results were analyzed by two investigators (D. Gao and C. Zhang) who were blinded to the results of in vivo NIRF imaging and tumor therapy.
For CD206 and F4/80 overlay staining, 4T1 tumor, muscle, liver, or spleen tissue slices were incubated with rat anti-mouse CD206 (BioLegend, San Diego, CA) and rabbit anti-mouse F4/80 (Abcam, Cambridge, MA) primary antibodies for 1 h at room temperature, and then visualized with Cy3- or FITC-conjugated secondary antibodies under a confocal microscope. For Ki67 staining, the tumor slices were incubated with rabbit anti-Ki67 antibody (Millipore, Billerica, MA). The slices were then visualized with Cy3-conjugated secondary antibody under a confocal microscope. After staining, 10 random views in the tumor slices were selected for the analyses. The murine CD206 expression level was calculated by measuring the integrated optical density of images of an equivalent area using a previously described method 23 (link). The tumor cell proliferation index was calculated as the percentage Ki67-positive cells 24 (link).