Validation of scRNA-seq results using flow cytometry and confocal laser microscopy. (A) Representative flow cytometry plots showing the expression of HLA-DR on PP-derived CD24⁺PDPN⁺ FB (left panel) and CD24⁻PDPN⁺ FB (right panel). (B) Quantification of HLA-DR^hi cells among CD24⁺PDPN⁺ FB (left panel) and CD24⁻PDPN⁺ FB (right panel). Dots represent individual samples; bars show the mean of data pooled from two (M-ILF), three (PP and SM-ILF), or four (SM, LP) tissue donors, and whiskers indicate SD. Pooled data are from four separate experiments. (C) Confocal laser microscopy images of large intestinal SM-ILF (left) and surrounding LP (right). Yellow boxes in the left panels highlight magnified areas. PDPN was chosen to highlight all FB, CD24, ADAMDEC1, CD34, and F3, the indicated FB subsets, and CD31 was chosen to highlight endothelial cells. The yellow arrows mark ADAMDEC1⁺PDPN⁺, CD34⁺PDPN⁺, and F3⁺PDPN⁺ FB, respectively. DAPI highlights all nucleated cells. Results are from one representative image of ≥3 replicates from different donors per staining that were stained in independent experiments.