Immunohistochemical Analysis of Liver Lipid A

Immunohistochemical analysis was performed as described elsewhere [21 (link)–23 (link)]. For this assay, livers were immediately fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 12 h, then soaked in 30% sucrose at 4°C for 24 h, and stored at -80°C until use. The livers were sliced at 7 or 10 μm thickness on a Microm HM-505 N cryostat. Staining was conducted by means of goat polyclonal antibodies against Lipid A of Enterobacteriaceae bacteria (cat. #: PA1-28903, Invitrogen, USA, 1:200), followed by probing with fluorescently labeled secondary antibodies (Abcam, USA). The slices were treated with the Phalloidin-iFluor 488 Reagent (cat. #: ab176753, Abcam), coverslipped with the Fluoro-shield mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI; cat. # F6057, Sigma-Aldrich, USA), and visualized under an AxioImager A1 microscope (Zeiss) with camera AxioCam MRc (Zeiss).