3 3 diaminobenzidine liquid substrate system

A High Sensitivity GLP-1 Active ELISA Kit (Cat# EZGLPHS-35K, Millipore, MA, USA) was used to measure GLP-1 in serum. Recombinant GLP-1 peptide (Human, Cat #SCP0153), a 3,3′-Diaminobenzidine Liquid Substrate System (Cat# D3939), an Alkaline Phosphatase Diethanolamine (ALP) Activity Kit (Cat# AP0100), Alizarin Red S (Cat# A5533), and a Masson Stain Kit (Cat# HT15) were purchased from Sigma-Aldrich (MO, USA). Primary antibodies were used to detect GLP-1 (Cat# ab22625, Abcam, MA, USA), RUNX2 (Cat# 12556, Cell Signaling Technology, MA, USA), MSX2 (Cat# ab69058, Abcam, MA, USA), SOX9 (Cat# 82630, Cell Signaling Technology, MA, USA), BMP2 (Cat# ab14933, Abcam, MA, USA), BMP4 (Cat# ab39973Abcam, MA, USA), and β-actin (Cat# 4970, Cell Signaling Technology, MA, USA) in immunohistochemical (IHC) or immunoblot assays. The secondary antibodies were horseradish peroxidase (HRP)-conjugated anti-rabbit antibodies (Cat# 7074, Cell Signaling Technology, MA, USA) or Alexa Fluor 594- or Alexa Fluor 488-conjugated anti-rabbit antibodies (Cat# R37119 or Cat# A27034, Thermo Fisher Scientific, NY, USA). Fetal bovine serum (FBS, Cat# 16000044), DMEM:F12 culture medium, penicillin, and streptomycin were from Gibco BRL (NY, USA).

Paraffin-embedded tissue sections were deparaffinized in xylene and rehydrated in a series of graded alcohols. After incubation of 3% H₂O₂ in PBS with 0.1% Triton X-100 at room temperature for 30 minutes, the sections were blocked with 5% bovine serum albumin (BSA) in PBS for 1 hour. The sections were incubated with a rabbit polyclonal anti-GALNT2 antibody (Sigma-Aldrich) at 1:200 in 1% BSA/PBS at 37ºC for 16 hours. After rinsing twice with PBS, the Super Sensitive Link-Label immunohistochemistry Detection System (BioGenex) was applied to tissue sections. Specific immunostaining was visualized with 3,3-diaminobenzidine liquid substrate system (Sigma-Aldrich). All sections were counterstained with hematoxylin and mounted with UltraKitt (J.T. Baker). For negative controls the primary antibodies were replaced with a control non-immune IgG at the same concentration. The immunoreactivity of GALNT2 was examined in a blinded manner by two independent investigators (Dr. Hsu WM and Dr. Jeng YM) without knowledge of clinical background of the patients. GALNT2 expression levels in NB tumors examined by immunohistochemical staining correlated well with those examined by Western blotting, as described in our previous study [22 (link)].

One hundred eighty-four tumor specimens collected before chemotherapy were fixed in formalin and embedded in paraffin. The expression of C1GALT1 was evaluated by using an avidin-biotin complex immunoperoxidase staining technique as described previously [37 (link)]. A monoclonal anti-C1GALT1 antibody (Santa Cruz) was used and signals were detected with Super Sensitive Link-Label immunohisto-chemistry Detection System (BioGenex). The specific staining was visualized with 3,3-diaminobenzidine liquid substrate system (Sigma) and counterstained with hematoxylin (Sigma).

Xenografts were paraffin-embedded and incubated with mouse anti-LMP1 monoclonal antibodies (1:400, Abcam) diluted with 5% bovine serum albumin/PBS for 12 h at 4 °C. After rinsing twice with PBS, Super Sensitive Link-Label Immunohistochemistry Detection System (BioGenex) was used and the specific immunostaining was visualized with 3,3-diaminobenzidine liquid substrate system (Sigma-Aldrich). All sections were counterstained with hematoxylin.

Paraffin-embedded HCC paired tissues were stained with anti-GALNT1 antibody, detected by Super Sensitive Link-label Immunohistochemistry Detection System (BioGenex), and visualized with 3,3-diaminobenzidine liquid substrate system (Sigma). Tissue sections were counterstained with haematoxylin for staining of nucleus. Negative control was done by replacing primary antibody with control IgG.

OxLDL (oxLDL, Serotec, UK) was used to stimulate smooth muscle cells. Immunohistochemical and western-blot antibodies used to detect TLR4, α-SMA, IL-1β, TNF-α, MCP-1 and MMP-2 were purchased from Abcam (MA, USA). 3, 3′ -Diaminobenzidine Liquid Substrate System was purchased from Sigma-Aldrich (MO, USA). Fetal bovine serum (FBS), F12: DMEM culture medium, penicillin and streptomycin were from Gibco BRL (Carlsbad, USA). The primary antibodies used were TLR4 (Abcam, USA), β-actin, NF-κB, phosphorylated-NF-κB, p38MAPK, phosphorylated-p38MAPK (p-p38MAPK), extracellular signal-regulated kinase 1/2 (ERK1/2), p-ERK1/2, c-Jun N-terninal kinase (JNK) and phosphorylated-JNK (Cell Signaling, MA, USA). HRP-conjunct antibody, Alexa 549- or Alexa 488- conjugated antibody (Cell Signaling, USA) was used as secondary antibody. Activation of TLR4 was blocked by Anti-mouse TLR4/MD-2 antibody (eBioscience, 16–9924, USA) and normal mouse IgG (eBioscience, USA) was used as negative control. Interleukin 1-β (IL-1β), tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein 1 (MCP-1) and matrix metalloproteinase-2 (MMP-2) ELISA kits were obtained from R&D Systems (R&D, USA).

These were performed essentially as described (16 ) unless otherwise specified. In brief, NGLs or glycolipids (50–150 pmol) were applied onto the HPTLC plates and developed with the solvent system CHCl₃/MeOH/0.5 m sodium acetate (25:25:8) for glycolipid extract of PC3 cells, and CHCl₃/MeOH/H₂O (60:35:8) for NGLs and human red cell-derived glycolipids. The plates were air-dried and blocked with 3% (w/v) BSA (Sigma A8577) in phosphate/buffered saline (10 mm phosphate, 2.7 mm potassium chloride, and 137 mm sodium chloride, pH 7.4) or HEPES-buffered saline (5 mm HEPES, pH 7.4, 150 mm NaCl, 5 mm CaCl₂) and overlaid with the following murine mAbs: F77 IgG3 at 20 μg/ml; IgG3 isotype control designated MG3-35 (Abcam) at 20 μg/ml or anti-H type 2 IgG1 designated BRIC231 (Abcam) at 1:100, followed by biotinylated anti-mouse immunoglobulins (Dako) at 1:200 dilution. Binding was detected by overlaying with streptavidin peroxidase (5 μg/ml) followed by color development using 3,3′-diaminobenzidine liquid substrate system (Sigma).