Fig. 3

Bacteria are transported to club cells. a An oblique section through the skin of a fish after overnight incubation in pHrodo Red labeled E. coli. The cytoplasm of two club cells is diffusely labeled (yellow arrowheads); other club cells do not have this diffuse pHrodo red fluorescence in their cytoplasm (red arrowheads). See Supplementary Movie 7. b A transverse section through the z-stack in a, showing one labeled club cell (yellow arrowhead). c.A schematic diagram of the slice in panel a, showing the different cell types. d Oblique section through the skin of another fish after overnight incubation in pHrodo Red labeled E. coli. Puncta are visible in goblet cells (arrowhead). e Localization of pHrodo Red labeled E. coli to perinuclear organelles of club cells (arrowheads). f, g Entry of a cell carrying pHrodo Red labeled E. coli into a club cell. f The cell (arrowhead) is initially adjacent to the club cell, but appears to have transferred some of its cytoplasm into the club cell. g After 30 min, the cell appears to be inside the cytoplasm of the club cell, which is now more intensely labeled. Localization of pHrodo red labeled S. aureus (h, i) and zymosan (j, k) within the epithelium, after transient incubation. l A neutrophil with bacteria, visualized by the EUB338 probe. Fish cells are labeled with SYTO 9 (green). This is a surface rendering of a z-stack, showing all focal planes. m A single plane of the stack in panel l, showing SYTO 9 label. The nucleus of the neutrophil (arrowhead) resembles the cell seen inside the club cell in Fig. 1e. n, p Two different focal planes of a region of the skin following in situ hybridization with the EUB338 probe. n At a deeper plane, several puncta are visible (white arrowhead), associated with a nucleus. One puncta is also associated with a second cell in a neighboring club cell (yellow arrowhead). o Transverse section through the club cell in n, with two puncta visible (arrowhead). p A shallower focal plane, showing the characteristic bi-lobed nucleus of a club cell (magenta arrowhead). q, r Transverse sections through the z-stack in p, showing labeled puncta associated with the deeper cell (arrowheads). s A rounded cell inside a club cell, labeled with probe to 16S rRNA. Scale bar = 10 µm