Fig. 7.

Intracellular uptake of fluorescein isothiocyanate (FITC)-lipopolysaccharide (LPS) in mouse jejunal epithelium in vivo. Mouse jejunal mucosa was exposed to luminal FITC-LPS solution (50 µg/ml) alone for 15 min (C), followed by to FITC-LPS with oleic acid (OA, 30 mM) plus taurocholate (TCA, 10 mM) (A, B, D, E) under isoflurane anesthesia. Localization of FITC-LPS was imaged using single-photon or two-photon confocal microscope. No FITC signal was observed in the villi with single-photon confocal imaging (A), whereas two-photon confocal imaging detected intracellular FITC signals in the presence of OA plus TCA (B) in the apical cytosol of villous cells by horizontal scanning (arrowheads) and in the bottom of villous cells by vertical scanning (red arrows) with unstained nucleus (white arrows). Luminal FITC-LPS alone showed no obvious intracellular staining of FITC-LPS with faint staining on the apical surface of villous cells (C). Addition of OA plus TCA to FITC-LPS solution rapidly stained intracellular space of villous cells with a negative paracellular space (D). Deeper scanning revealed cytosolic staining in the bottom of villous cells with negative nuclei (white arrows in D and E). Exposure to luminal a cell-impermeant red fluorescence dye SNARF-5F with OA plus TCA failed to stain the intracellular space. Internal bar = 50 µm.