Visualizing Intestinal Bacterial Colonization

Formalin-fixed paraffin-embedded intestinal tissue sections (5 µm) were initially treated with lysis buffer for 1 h at 37 °C and then hybridized at 51 °C with a 20 bp bacteria-specific probe (EUB 338: GCTGCCTCCCGTAGGAGT) to visualize and quantify the proximity of luminal bacterial colonies and the corresponding antigen load on the epithelium. Following the overnight hybridization, the intestinal sections were counter-stained with 4,6-diamidino-2-phenylindole (DAPI, Dihydrochloride, Cat #: D1306 by Invitrogen) as previously described [40 (link)] for visualization of cell nuclei. Images were obtained and analyzed using a Leica DM8 i SPE confocal microscope.