Intestinal tissues were fixed in 4% paraformaldehyde, rehydrated in 20% sucrose, and frozen in OCT media (Sakura). Tissues were cut into 7-8μm sections and treated with ice cold acetone. Sections were treated with biotin-avidin blocking reagent when necessary (Vector labs) and stained with the following biotinylated or directly conjugated antibodies: CD8β (YTS156.7.7, Biolegend), CD4 (RM4-5, eBioscience), CD103 (M290, BD Biosciences), CD90.1 (HIS5.1, eBioscience), Epcam (G8.8, Biolegend), CD11c (HL3, BD Biosciences), B220 (RA3-6B2, eBioscience). Rabbit anti-Yersinia pseudotuberculosis (ab26120, Abcam) and anti-rabbit Dylight 649 (ab96926, Abcam) were used to stain for bacterial antigens. Stained slides were mounted with Prolong Gold antifade reagent (Invitrogen), imaged using a Nikon 90i, and analyzed using Adobe Photoshop software.
The number of OT-I cells/villus was determined by sectioning a ‘Swiss roll’52 (link) of the distal third of the small intesine. Five or more sections/mouse that were at least 400μm apart were stained and imaged. A villus and the underlying submucosa and muscularis were considered a single villus, and the number of OT-I cells in each region was determined. The number of OT-I cells/villus were binned and plotted as the percentage of villi containing a given range of OT-I cells.
The number of OT-I cells/villus was determined by sectioning a ‘Swiss roll’52 (link) of the distal third of the small intesine. Five or more sections/mouse that were at least 400μm apart were stained and imaged. A villus and the underlying submucosa and muscularis were considered a single villus, and the number of OT-I cells in each region was determined. The number of OT-I cells/villus were binned and plotted as the percentage of villi containing a given range of OT-I cells.