Ep1347y

Each antibody was validated using conventional immunohistochemistry and monoplex immunofluorescence staining in conjunction with the corresponding opal fluorophore and the spectral DAPI counterstain. The antibodies were tested at three different dilutions, starting with manufacturer recommended dilution (MRD), MRD/2, and MRD/4 with 1/100 tyramide to select the optimal concentration of antibody capable of generating the best signal. The signal was then optimized through different tyramide titrations until generation of the best exposure time ranging from 5 to 25ms. Reproducibility was evaluated using: a positive control of each antibody with DAPI and a DAPI-alone slide. The negative controls included one unstained slide for auto-fluorescence compensation, a tyramide-alone slide treated with hydrogen peroxide for endogenous peroxidase masking, and finally a secondary antibody with tyramide slide. The following antibodies were used in the multiplex panel analysis: S100B Leica NCL-L-S100167 1/100 pH6 with Tyr480 1/200; pSTAT3 Cell Signaling Tyr705 D3A7 XP 1/200 pH 6 with Tyr520 1:150; CD68 Agilent PG-M1 1:50 pH 9 with Tyr570 1:150; CD3 Agilent clone F7.2.38 1:50 with pH 6 buffer for antigen retrieval with Tyr620 1:300; CD163 Abcam EPR19518 1:500 pH 9 with Tyr690 1:100; and CD11c Abcam EP1347Y 1:300 pH 9 with Tyr780 1:100.

Frozen samples were cut into10-μl sections by using a cryostat (Microm HM550, Thermo Fisher Scientific), fixed in 10% formalin, and stained with hematoxylin and eosin (HE). For DC staining with anti-CD11c (EP1347Y, Abcam), the fixed sections were incubated in a 0.05% Tween 20/citrate buffer (pH 6.0) solution at 60°C overnight to retrieve antigenicity according to the manufacture’s instructions. Then, the sections were stained with anti-CD11c followed by Alexa Fluor 647 anti-rabbit IgG and counterstained with Hoechst 33258 (Dojindo, Kumamoto, Japan). Resultant sections were examined by using a confocal laser microscope (Fluoview FV10i, Olympus, Tokyo, Japan) or a fluorescence microscope (Biozero, Keyence, Osaka, Japan).

Japanese NAFLD/NASH patients who had sustained liver dysfunction, dyslipidemia, and insulin resistance were recruited at Yamaguchi University Hospital and Yokohama City University Hospital. We measured body mass index and serum parameters according to the standard procedures. LSM and CAP values were determined by FibroScan (Echosens). Liver samples were obtained by ultrasound-guided liver biopsy to evaluate liver histology. NAS score and fibrosis stage were assessed according to the NASH clinical research network scoring system. Formalin-fixed and paraffin-embedded liver specimens were stained with antibodies against CD68 (M0876; Dako), CD11c (EP1347Y, Abcam), and CTSD.