3 30 diaminobenzidine tetrahydrochloride dab

The sections at each period were deparaffinized and soaked in 0.3% hydrogen peroxide. Cluster of differentiation 68 (CD68) was used as a marker of macrophage-like type A synoviocytes [24 (link)]. Endogenous peroxidase was inactivated with 3% H₂O₂ in PBS for 20 min at room temperature. The slides were incubated with mouse anti-rat CD68 antibody (MCA341 R, AbD Serotec, Raleigh, NC, USA; dilution, 1:400) for 24 h at 4 °C. Goat anti-mouse immunoglobulin (IgG) (Nichirei, Tokyo, Japan) for CD68 was used as the secondary antibody and incubated for 30 min at room temperature. The final detection step was carried out using 3,30-diaminobenzidine tetrahydrochloride (DAB) (Sigma, St. Louis, MO, USA) in 0.1 M imidazole and 0.03% H₂O₂ as the chromogen. Counterstaining was performed using Carazzi’s hematoxylin. For the negative control, normal mouse IgG (Dako, Copenhagen, Denmark) was used as the primary antibody. All slides were stained in one session. CD68-positive cells were enumerated at the posterior capsule including joint space and synovium [25 (link)].

The recombinant plasmids pET30a-H (GenBank Accession No. X74443), pCAGGS-F (GenBank Accession No. X74443) and vector pCMV-HA were provided by the Lanzhou Veterinary Research Institute of Chinese Academy of Agricultural Sciences and were used to construct eukaryotic expressing plasmids. CHO-K1 cells were obtained from Shanghai Institutes for Biological Sciences (SIBS). E. coli DH5α, T4 DNA ligase and all restriction enzymes were purchased from TaKaRa. The QIAprep® spin miniprep kit was from QIAGENE. F12 K, G418, OPTI-MEM medium and Lipofectamine3000 were products of Invitrogen. FCS was purchased from Gibco BRL Life Tech. Co-IP kit was purchased from Thermo Fisher. Mouse anti-HA monoclonal antibody, isotype control antibody, lexa Fluor 488/HRP-conjugated anti-mouse IgG and 3,30-diaminobenzidine tetrahydrochloride (DAB) were purchased from Sigma. Immobilon-P transfer membranes were purchased from Millipore. CM5 sensor chip, amine coupling kit and all solutions were also purchased from GE Healthcare.

Samples including crude antigens (15 μg/lane), ES antigens (15 μg/lane) and the rTspst proteins (3 μg/lane) were separated by SDS–PAGE and then transferred onto nitrocellulose membranes (Millipore, USA) using a trans-blot SD transfer cell (Bio-Rad, USA) [27 (link)]. The membranes were cut into strips, blocked with 5% skimmed milk in Tris-Buffered Saline with Tween-20 (TBST) at 37°C for 1 h, and incubated at 37°C for 1 h with 1:100 dilutions of different mouse sera (anti-rTspst serum, serum from mice infected T. spiralis at 30 dpi and normal mouse serum). After washing, the strips were incubated at 37°C for 1 h with HRP-conjugated goat anti-mouse IgG (1:5000 dilution; Southern Biotechnology, USA), and finally with 3, 30-diaminobenzidine tetrahydrochloride (DAB; Sigma).