Visualization of enterocyte efferocytosis in the neonatal intestine in vivo. Immunostaining and TEM of small intestinal tissue sections of S. Typhimurium–infected neonate mice at day 4 p.i. (A and B) Immunostaining for cleaved caspase 3 (cl. Casp. 3, red), CD36 (orange), and S. Typhimurium (green). Focal CD36 expression by epithelial cells of the opposite villus (A) or by neighboring cells within the epithelial cell layer (B) of cleaved caspase 3–positive and in A also S. Typhimurium–positive IECs. Counterstaining with DAPI (blue). Bar, 20 μm. (C) Number of cleaved caspase 3 (cl. Casp 3)–positive cells with an adjacent CD36 signal among all cl. Casp 3–positive cells. (D) Immunostaining for CD36 (orange), TUNEL (white), and cleaved caspase 3 (cl. Casp3, red). Counterstaining with DAPI (blue). Bar, 20 μm (overview image) and 10 μm (single-color channels). (E) Number of TUNEL-positive cells with an adjacent CD36 signal among all TUNEL-positive cells. (C and E) 21 image fields with the size of 312.35 × 250.61 µm of small intestinal tissue sections from four individual S. Typhimurium–infected neonatal animals at day 4 p.i. were analyzed. (F) TEM images showing long membrane protrusions extending from the apical plasma membrane of the epithelium (arrows) engulfing luminal cell debris. Arrows in the left inset in panel i indicate the septum of a dividing S. Typhimurium (S) within an efferosome (Ef). Panel ii is a higher-resolution image of the area indicated in panel i. Two bacteria (S) are situated in the lumen within cell debris (iii). Panel iii was imaged on a serial section through the same tissue block. Bar, i = 5 μm, ii and iii = 1 μm. (G) Immunostaining for CD36 (orange) and S. Typhimurium (green). Counterstaining with DAPI (blue). Bar, 10 μm.