Horseradish peroxidase conjugated streptavidin

MaxiSorp plates were coated with full-length OPN, the N-terminal part of OPN (Ile¹-Arg¹⁵²) or the C-terminal part of OPN (Ser¹⁵³-Asn²⁹⁸) (3 µg/ml) in phosphate buffered saline (PBS) overnight at 4°C. Subsequently, the wells were blocked with 2% Tris-Tween buffer (2% Tween 20, 10 mM of Tris-HCl, 1 M of NaCl, 10 mM of CaCl₂, pH 7.4) for 1 h at room temperature. A reaction mixture (0.1 ml) containing 10 µM biotinyl-TVQQEL-OH or 5-(Biotinamido)pentylamine, and 0, 0.25, 0.5, 1, 2, 3, 5 µg/ml TG2 in 40 mM of Tris-HCl (pH 8.3), 140 mM NaCl, 10 mM CaCl₂, 5 mM dithioerythritol was added to the plates and incubated for 3 h at 37°C. Biotinyl-TVQQEL-OH and 5-(Biotinamido)pentylamine cross-linked to OPN were detected by incubation with horseradish peroxidase-conjugated streptavidin (Dako) (diluted 1∶8000) for 1 h at 37°C. Color development was obtained with TMB-one substrate (Kem-En-Tec) and the reaction was stopped by addition of 0.2 M H₂SO₄. Color intensity was measured at 450 nm using an ELISA reader.

WT and SIRT1 TG mice (8-week-old) were treated with 1 mg/kg Pam3CSK4 for 24 h and then killed. Tissues were fixed through a systemic perfusion, and aortas were dissected carefully and immersed in 10% buffered formalin. Aortic segments were incubated with the anti-rat Mac-1 monoclonal antibody and biotinylated goat anti-mouse IgG, and then reacted with horseradish peroxidase-conjugated streptavidin (Dako). Six to 10 images of each field were captured at various focal lengths using an automatically regulated Z-stepper and Image-Pro4.5J software (Planetron Co., Tokyo, Japan).
Hearts were fixed with 4% paraformaldehyde, dissected, and the proximal half was mounted in OCT compound (Sakura Finetek Inc., Torrance, CA) to prepare the aortic sinus tissue sections. Five-micrometer-thick cross-sections were cut until the aortic sinus appeared. Afterwards, every other section was collected on a slide until the valve cusps were no longer visible. Sections of the aortic sinus were stained with oil red O (Sigma Chemical Co.) to visualize the atherosclerotic lesions. The lesion area was quantified using Carl Zeiss Imaging Systems (AxioVision LE version 4.5, Carl Zeiss Inc., Jena, Germany).

Primary antibodies against Akt2 (#3063), pAkt-Ser473 (#9271), pAkt-Thr308 (#9275), pAMPKα-Thr172 (#2531), pACC-Ser79/212 (#3661), pTBC1D4-Ser588 (mouse: Ser595) (#8730), and pTBC1D4-Thr642 (mouse: Thr649) (#8881) were from Cell Signaling Technology (Danvers, MA, USA) Antibody against pTBC1D1-Ser231 (#NRG-1848963) was from Millipore (Burlington, MA, USA), AMPKα2 antibody (#SC-19131) and Hexokinase II were from Santa Cruz (Dallas, TX, USA)(#SC-6521) while GLUT4 antibody (#PA1-1065) was from Thermo Fisher Scientific (Waltham, MA, USA). ACC protein was detected using horseradish peroxidase-conjugated streptavidin from Dako (Glostrup, Denmark), (#P0397). TBC1D1 and TBC1D4 protein as well as phosphorylation of TBC1D4-Ser711 were detected using antibodies as previously described [31 (link),30 (link)].

The binding of native and Fe(II)-IVIG to FAS was determined by a dot blot assay on nitrocellulose membrane (Millipore, Bedford, UK). Recombinant FAS (Enzo Life Sciences AG) in PBS was dotted at 10 μg/mL on nitrocellulose filter membranes. Dots were blocked with PBS containing 0.05% Tween 20 (Sigma-Aldrich) and 5% bovine serum albumin (BSA; Sigma-Aldrich) and incubated with IVIG preparations (20 mg/mL) overnight at 4°C. The membranes were washed with PBS containing 0.05% Tween 20. Quantification of FAS-specific IVIG antibodies was done by incubation with anti-human IgG1 (Biotin-SP-conjugated AffiniPure goat anti-human IgG + IgM, Jackson ImmunoResearch, West Grove PA, USA, 1:20'000 in PBS/T) and subsequent detection with horseradish peroxidase-conjugated streptavidin (DAKO, Glostrup, Denmark, 1:3'000 in PBS/T). Membranes were soaked in enhanced chemiluminescence detection reagent (Millipore; Burlington MA, USA) and visualized using the luminescent image analyzer LI-COR® Odyssey (Lincoln NE, USA).

Mouse lungs were perfused with 10 mL of saline (through the right ventricle), harvested, and fixed overnight with 4% paraformaldehyde. Fixed samples were embedded in Tissue-Tek® O.C.T. compound (SAKURA Finetek) and serially sectioned (3 μm thickness). The tissues were incubated in 0.3% H₂O₂/methanol for 30 min to block endogenous peroxidase activity. The sections used for CD31 staining were heat-treated in 0.01 M citrate buffer (pH 6.0) for antigen retrieval before peroxidase blocking. Tissues were blocked with 5% goat serum and incubated with appropriate primary antibodies overnight at 4 °C, followed by secondary antibodies for 60 min at room temperature. The following primary antibodies were used: rabbit anti-mouse BLT2 (5 μg/mL; generated in our laboratory by immunizing a rabbit with a BLT2 C-terminal peptide); rabbit anti-proSP-C (1:1000; Millipore); hamster anti-T1α (1:2000; BioLegend); rabbit anti-CD31 (1:50; abcam), rabbit IgG (5 μg/mL; DAKO); and hamster IgG (1:2000; BioLegend). The secondary antibodies were biotinylated anti-rabbit goat IgG (1:300; DAKO) and biotinylated anti-hamster goat IgG (1:200; Vector Laboratories). Tissues were labeled with horseradish peroxidase-conjugated streptavidin (DAKO), and the signals were detected by incubating with diaminobenzidine. Images were captured under a microscope (Keyence BZ9000).