ALO medulla is made up of FRCs. (A) UMAP plot of ALO scRNAseq data highlighting the medullary FRC cluster. (B) Dot plot showing the relative gene expression of genes used to identify and characterize the FRC cluster. (C–G) Confocal (C and D) and DIC (E) micrographs of an ALO excised from an adult Tg(pdgfrb:EGFP)^ncv22 transgenic zebrafish with green fluorescent FRCs. Panel C shows an ALO overview image, and panels D and E show higher magnification images of the yellow boxed area in panel C. Panel F shows an extended depth-of-focus image with DIC, and panel G shows a higher magnification image of the boxed region in panel F, showing FRCs in white and pericytes surrounding a blood vessel (also labeled by the transgene) pseudocolored magenta. (H and I) Confocal micrographs of the ALO of an adult zebrafish subjected to HCR for spock3 (magenta), with DAPI counterstain shown in white. Panel I shows a higher magnification image of the boxed area in panel H. (J) Successive images from a time-lapse DIC video micrograph of an immune cell (pseudocolored magenta) migrating along the FRC network. Images are selected frames from Video 6. (K–O) Transmission electron micrographs of the ALO medulla. Panels K and L show immune cells (pseudocolored magenta) in close apposition to FRCs (pseudocolored blue). Panels M and N show an immune cell (magenta arrow) closely apposed to an FRC body (black arrow) embedded in the matrix surrounding a lymphatic vessel. Panel N shows a magnified image of the boxed region in panel M, with an immune process (magenta arrow) extending across the FRC (yellow asterisks). Panel O shows three FRC bodies (black arrows) surrounding a lymphatic vessel (lymphatic endothelium noted with magenta arrows), with FRC lamellar extensions noted with green arrows and the thick matrix layer surrounding the vessel noted with magenta asterisks. Transgenic imaging, n = 7; HCR, n = 4; TEM, n = 2. Scale bars = 100 µm (C), 20 µm (D and I), 50 µm (F–H), 10 µm (J), 5 µm (K and L), 2 µm (M), 500 nm (N), 4 µm (O).