Fig 1: Biodistribution of CpG by C-agarose Gel and Cell Transfection Assay(A) The levels of CpG in different organs from 4T1 tumor-bearing mice were examined 1 week after naked agarose+Cy3-CpG or C-agarose+Cy3-CpG gel administration. (B) Frozen sections of TDLN from tumor-bearing mice treated with C-agarose+Cy3-CpG gel implantation were stained with a Siglec-1 antibody (red, Cy3-CpG; green, Siglec-1; blue, DAPI nuclear staining). Scale bar, 100 µm for 100× or 20 µm for 400×. (C) Lymph node cells from tumor-bearing mice treated with agarose+Cy3-CpG or C-agarose+Cy3-CpG were examined by flow cytometry. (D) The levels of CpG in Siglec-1+ macrophages and Siglec-1- cells were quantified by fluorescence intensity. (E and F) In vitro results of C-agarose+CpG transfection into lymph node sinus macrophages were examined by confocal microscopy (E) and quantified by a flow cytometer (F). Siglec1 siRNA, mismatched siRNA (m-siRNA), control plasmid, or Siglec-1 plasmid was transfected into Siglec-1+ macrophages via Lipofectamine 2000 48 h before the addition of C-agarose+Cy3-CpG complex. Fluorescence images (G) and flow cytometry (H) results of above treated Siglec-1+ macrophages (G and H) (red, Cy3-CpG; blue, DAPI nuclear staining). Scale bar, 100 µm (E and G). n = 10 mice per group. Data are representative results from five independent experiments and are expressed as the mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001. Lipo, Lipofectamine 2000; MF, macrophages; TDLN, tumor-draining lymph node.