Extravidin horseradish peroxidase

Sections were immunostained for CD31, to indicate neovascularization, by incubating them with primary mouse anti-CD31/PECAM-1 monoclonal antibody (dilution, 1:150; cat. no. NB100-1642, Novus Biologicals, LLC, Littleton, CO, USA) and biotinylated goat anti-mouse IgG (dilution, 1:150; cat. no. NBP1-97590; Novus Biologicals, LLC). The sections were then incubated with ExtrAvidin-horseradish peroxidase (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Aminoethylcarbazole was used as a chromogenic substrate using an Aminoethylcarbazole Staining kit (Sigma-Aldrich; Merck KGaA). Microphotographs were captured, and 20 random fields of 3 stained sections (>4 fields/section) from each group were observed at ×40 magnification from central healing areas for semiquantitative analysis of microvessel density. Negative control sections were incubated with PBS instead of the primary antibody.

Serial 5 μm paraffin sections were de-waxed, rehydrated and the endogenous peroxidase activity inhibited as previously described (Scholtes et al., 2012 (link)). After rinsing, sections were heated in 10 mM citrate buffer (pH 6.0) for 2 periods of 6 min. Following blocking with 20% (v/v) goat serum in PBS, sections were incubated overnight at 4 °C with antibodies against human ADAMTS-1 (Santa Cruz, sc-25581), ADAMTS-4 (Thermo Fisher Scientific, PA1-1749) and ADAMTS-5 (Santa Cruz, sc-134952), or an antibody against human macrophage CD68 (Dako, Cambridgeshire, UK, PGM1) or human α-smooth muscle actin (Dako, 1A4) in 1% (w/v) BSA in PBS. After overnight incubation, sections were washed and incubated with an appropriate biotinylated secondary antibody, followed by incubation with extravidin horseradish peroxidase (Sigma–Aldrich, St Louis, MO) and stained with 3,3-diaminobenzidine (DAB) (Sigma–Aldrich). The sections were then counterstained with hematoxylin, mounted in DPX mountant and visualized on a light microscope (Leica).

Circulating IgA1 and Gd-IgA1 were detected by ELISA as previously described.¹⁵ (link) Briefly, high-binding MaxiSorp 96-well plates (Nalge Nunc, Rochester, NY) were coated with F(ab’) 2 fragment of goat antihuman IgA (Jackson Immuno-Research Labs, West Grove, PA). After overnight incubation at 4 °C, the coated plates were washed and blocked with 1% bovine serum albumin (Sigma Chemical Company, St Louis, MO). Then, diluted serum samples and standards were added. After incubating for 1 hour, the level of IgA1 was determined by incubation with horseradish peroxidase–labeled mouse antihuman IgA1 antibody. As for Gd-IgA1, samples were treated with sialidase A and biotin-labeled helix aspersa agglutinin after incubating. After another incubation, the plates were further incubated with horseradish peroxidase-ExtrAvidin (Sigma). The plates were then developed with the peroxidase chromogenic substrate o-phenylenediamine-hydrogen peroxide (Sigma). The color reaction was stopped with 1 M sulfuric acid, and the absorbance was measured at 490 nm with an EL312 Bio-Kinetics microplate reader (Bio-Tek Instruments Inc., Winooski, VT).