3 3 diaminobenzidine (dab)

TLR ligands (ApoTech) and TLRgrade™ LPS from E. coli serotype O55:B5 (Enzo Life Science, Inc.) were used (Fig. 2). In addition, polymyxin B (Wako) and APDC (Dojindo) were dissolved in water and filter-sterilised. For electron microscopy, DAB and 3% (v/v) hydrogen peroxide (Muto Pure Chemicals) were used. To inhibit neutrophil elastase and MPO, Sivelestat and 4-ABAH were purchased from Cayman and used for the neutrophil inhibition assay as described above.

Embryos were fixed with 4% paraformaldehyde in PBS overnight at room temperature. The primary antibody (rat anti-GFP IgG, Nacalai tesque or mouse anti-HuC IgG, Santa Cruz Biotechnology) and the secondary antibody (biotin-conjugated anti-rat IgG or biotin-conjugated anti-mouse IgG, VECTOR) were used at a 1∶1000 dilution. Staining was done by using VECTASTAIN ABC kit (VECTOR) with DAB (MUTO PURE CHEMICALS) or Alexa Fluor 488 tyramide (Invitrogen). In the experiment that detects HuC-positive enteric neurons, immnostaining was performed on cryostat sections made from frozen samples mounted in O.C.T. (Sakura Finetek Japan).

Chicken anti‐NeuGc monoclonal IgG Abs (HU/Ch2‐7; Pharma Foods International Co., Ltd., Kyoto, Japan) were used for the detection of NeuGc Ag in the hepatectomized liver tissues.¹⁰, ¹¹ HU/Ch2‐7, which reacted strongly with NeuGc Ag, was specifically directed toward an antigenic epitope containing NeuGc.¹¹The sections were incubated with HU/Ch2‐7 in phosphate‐buffered saline (PBS) overnight. Biotin‐conjugated goat anti‐chicken IgY (H&L) polyclonal Ab (Abcam, Cambridge, USA) in PBS was added and incubated for 30 min. The sections were incubated with horseradish peroxidase‐conjugated streptavidin (Histofine SAB‐PO Kit; Nichirei Biosciences, Tokyo, Japan) for 30 min. Peroxidase activity was analyzed by staining the sections with 3,3′‐diaminobenzidine (Muto Pure Chemicals, Tokyo, Japan) for 10 min.¹² The sections were examined and photographed at 400X magnification using a BZ‐9000 microscope (Keyence, Osaka, Japan). The quantification of fluorescence intensity was performed using the Hybrid cell count BZ‐H2C software (Keyence). Based on the percentage of immunoreactive cells within the microscopic field of view, NeuGc Ag levels were classified into two groups: negative (0%) and positive (>0%) group. Four of the 66 initial hepatectomy samples were excluded because the quantification of the immunostaining Ag was impossible owing to poor storage conditions.