Goat anti mouse igg

Isotyping of the MAbs was performed using a Monoclonal Antibody Isotyping Kit (Bio-Rad, Hercules, CA, USA). The affinities of each MAb were measured using ELISA, as described previously [45 (link)]. In brief, the ELISA plate was coated with purified H9N2 HA protein (20 ng/well) overnight at 4 °C. MAbs were twofold serially diluted, starting at 1 mg/ml, and added to the plate. Incubation was carried out for 1 h at 37 °C. Then, goat anti-mouse IgG (Novus, St Charles, MO USA) was diluted 10,000 times and added as the secondary antibody. Incubation was carried out for 30 min at 37 °C. The color reaction was performed using the 3, 3′, 5, 5′-tetramethylbenzidine (TMB) reagent (KPL, Gaithersburg, MD, USA). After 10 min, the color reaction was stopped using the terminating reagent (KPL). Between each step, phosphate-buffered saline (PBS) with Tween 20 (PBST) was used to wash the plate five times. An ELISA plate reader (Bio-Rad) was read used to read the optical density (OD) at 450 nm, and the antibody’s affinity was estimated as the minimum concentration of the MAb required to provide a positive reaction. The variable genes of the heavy or light chains of the MAbs were sequenced by Sino Biological.

After examination by micro-CT, the samples were fixed in 4% PFA and then decalcified in 10% (w/v) EDTA (pH = 7.0) for 2 months at room temperature. Next, they were dehydrated and embedded in paraffin wax, sectioned into 6-μm slices and stained with H&E, toluidine blue, safranin O/fast green, and Sirius red according to the manufacturer’s protocols. Collagen II immunohistochemical staining was performed by immersing the sections into 0.25% pepsin (Abcam, United States) at 37°C for 20 min and blocking them in 10% goat serum for 1 h. After antigen retrieval, the slices were incubated with primary antibodies against collagen II (1:200; Developmental Studies Hybridoma Bank, United States) at 4°C overnight. After washing with PBS, they were incubated with goat anti-mouse IgG (1:200; Cat# NB7539; Novus) for 1 h. Finally, the sections were stained with Tris-HCl buffer containing 0.05% DAB and 0.005% hydrogen peroxide, and the nuclei were stained with hematoxylin. Photomicrographs were acquired using a Nikon microscope (Japan).
To evaluate the progress of subchondral bone reconstruction and cartilage repair, sections from three knees at 3 and 6 months per group (each sample represented three tissue sections) were blindly scored by three independent observers according to an established scoring system.