Flow cytometry and histological analyses of intestinal FB confirm heterogeneity between sites. (A) Violin plots showing the expression of the indicated genes by intestinal FB pooled from five (PP and SM-ILF), three (ileal and large intestinal LP and M-ILF), and one (ileal and large intestinal SM) samples. (B) Representative flow cytometry plots showing the gating of CD24⁺ FB within the total pool of PDPN⁺ FB and the gating of CD34⁺ and F3⁺ FB among CD24⁻ FB. Cells were pregated on live, CD235ab⁻EpCAM⁻CD45⁻CD31⁻PDPN⁺ cells. (C) Mean abundance (±SD) of CD24⁺ FB among total PDPN⁺ FB (upper left) and CD34⁻F3⁻ DN (upper right), CD34⁺ FB (lower left), and F3⁺ FB (lower right) among CD24⁻ FB. Each symbol represents one sample of two (M-ILF), three (PP and SM-ILF), or four (ileal and large intestinal SM and LP) tissue donors. Pooled data are from four separate experiments. (D) Confocal laser microscopy images of ileal GALT and surrounding LP. Yellow boxes in left panels highlight magnified areas. PDPN was chosen to highlight all FB, CD24, ADAMDEC1, CD34, and F3, the indicated FB subsets, and CD31 to mark endothelial cells. Yellow arrows mark ADAMDEC1⁺PDPN⁺, CD34⁺PDPN⁺, and F3⁺PDPN⁺ double positive FB, respectively. Images are representative of ≥3 replicates from different donors per staining that were stained in three or four independent experiments. DN, double negative.