Anti human cd68

For immunofluorescence, tissues were immediately isolated, fixed for 2 h with 4% paraformaldehyde in PBS at 4 °C and soaked in 30% sucrose in PBS overnight at 4 °C. Tissues were then embedded in Tissue-Tek OCT blocks (Sakura) and frozen in liquid nitrogen. The frozen samples were sectioned at a thickness of 10 μm by cryostat (Leica, CM3050S). The following antibodies were used: anti-mouse CD3ε (BioLegend, clone 145-2C11), anti-mouse B220 (eBioscience, clone RA3-6B2) and anti-mouse CD11c (SouthernBiotech, polyclonal). Tissue sections were counterstained with DAPI (Sigma-Aldrich) and mounted with Fluoromount-G antifade reagent (SouthernBiotech).
For human tissue staining, tissues were freshly isolated, embedded in Tissue-Tek OCT blocks (Sakura) or SCEM (SECTION-LAB) and frozen in liquid nitrogen. The frozen samples were sectioned at a thickness of 10 μm by cryostat (Leica, CM3050S). The following antibodies were used for immunohistochemistry staining: anti-human CD68 (eBioscience, clone 815CU17), anti-human CD19 (Abcam, clone EPR5906) and anti-human IgA (SouthernBiotech, polyclonal). Fluorescence images were obtained using a BZ-X700 fluorescence microscope (Keyence).

Fetal bovine serum (FBS), anti-cadherin 11 antibody, and TRIzol were purchased from Invitrogen (Carlsbad, CA, USA); anti-ERK, phosphorylated ERK (P-ERK), p38, P-p38 antibodies and anti-fibroblast marker (ER-TR7), A/G Plus-agarose, scrambled control, validated EPCR, PC, and sPLA₂V small interfering RNAs (siRNAs) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); recombinant APC from Eli Lilly and Company (Indianapolis, IN, USA); RiboCellin Transfection Reagents from BioCellChallenge (Paris, France); anti-β-actin and PC/APC antibodies and recombinant TNF-α from Sigma-Aldrich (St. Louis, MO, USA); anti-human CD68 from eBioscience (San Diego, CA, USA); human IL-1β, IL-6, and IL-8 enzyme-linked immunosorbent assay (ELISA) DuoSet from R&D Systems (Minneapolis, MN, USA); anti-nuclear factor-kappa B (anti-NF-κB) p65 subunit antibody from Chemicon International (Temecula, CA, USA); recombinant sPLA₂V from Abcam (Cambridge, MA, USA); anti-human sPLA₂V antibody from Cayman (Ann Arbor, MI, USA); NE-PER nuclear extraction reagents from Pierce Chemical Co. (Rockford, IL, USA). RCR252 (EPCR blocking antibody) and RCR92 (EPCR non-blocking antibody) were kindly provided by Kenji Fukudome (Saga Medical School, Saga, Japan).