Anti laminin l9393

The ileum was harvested at day 9 p.i., fixed in 2% paraformaldehyde, rehydrated in 20% sucrose in PBS, and flash frozen in optimal cutting temperature (OCT) media (Sakura Finetek, Torrance, CA, USA). The tissue sections were cut at a thickness of 7–8 μm and treated with ice cold acetone before storage at −80°C. Tissue sections were treated with avidin-biotin blocking reagents (Vector Laboratories, Newark, CA, USA) and stained with the following reagents: anti-CD45.2-biotin (104; Invitrogen), anti-CD90.1-APC (HIS51; Invitrogen), anti-laminin (L9393; Abcam, Cambridge, United Kingdom), anti-GFP-AF488 (Thermo Fisher Scientific), and anti-EpCam-AF647/BUV421 (G8.8; Biolegend), followed by Streptavidin-AF555 (Thermo Fisher Scientific), anti-rabbit AF488 (Thermo Fisher Scientific), and 1 μg/ml DAPI (Millipore Sigma). Stained slides were mounted with Prolong Gold antifade reagent, imaged using a Keyence fluorescence BZ-X series microscope (Osaka, Osaka, Japan), and analyzed using the Adobe Photoshop software. For the quantification of cells in lymphocyte clusters, the cells were counted in multiple sections for each mouse for > 700 total cells/mouse and > 200 cells in lymphocyte clusters/mouse. For examining the distance of LP Trm cells from the epithelium, the distance between the surface of each cell and the base of the nearest epithelial cell was determined for 150–360 cells/mouse.