Horseradish peroxidase conjugated goat anti mouse igg

The potential C1q binding peptide P^TsCRT (amino acid sequence: DLEDFNSDTPYRIMFGPDICGPEKR) derived from TsCRT^Δ was synthesized and its ability to competitively inhibit C1q binding to IgG was investigated using enzyme-linked immunosorbent assay (ELISA). First, 96-well plates were coated with human IgG (3 µg/ml, Abcam, UK) or the same amount of BSA as control in 100 µl/well of coating buffer overnight at 4°C. Wells were washed with PBST (136.75 mM NaCl, 2.68 mM KCl, 30.25 mM NaH2PO4, 1.76 mM KH2PO4, 0.05% Tween 20, pH 7.4) 3 times and then blocked with 200 µl 3% BSA in PBS at 37°C for 1h. Then, 100 µL of C1q (1 µg) pre-incubated with different doses of TsCRT^Δ (0, 4, 8, 16, 32, and 64 µg) was added to each well and incubated for 2h at 37°C. After washing the wells, anti-C1qA mAb (1:10,000, Abcam, Cambridge, UK) and horseradish peroxidase–conjugated goat anti-mouse IgG (1:10,000, ZSGB-BIO, Beijing, China) was sequentially added to the wells followed by incubation for 1h at 37°C. Next, TMB was added (100 µL/well, BD ebioscience, California, USA) as substrate and the absorbance value at 450 nm was measured with an ELISA reader (Thermo Fisher Scientific, Waltham, MA USA).

The osteosarcoma tumor samples from mice were fixed with 10% formalin for 1 day, paraffin-embedded and sectioned at 4 μm thickness. The sections were dewaxed, rehydrated, blocked with 5% bovine serum albumin (BSA), and incubated with an anti-Ki67 antibody (A2094, Abclonal, Wuhan, China) at 4°C for 16 h. The sections were then washed three times with PBS, incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (ZSGB-BIO, Beijing, China) at 37°C for 1 h, and then counterstained with hematoxylin solution. The procedure described above has been described in a previous study [20 (link)]. The intensity of anti-Ki67 staining was analyzed using a scanner control software (Pannoramic MIDI II, 3DHISTECH, Budapest, Hungary).