Goat anti mouse igg

At 12 and 28 weeks after the last immunization, antigen-specific immunoglobulin IgG, IgG1, and IgG2c in sera were detected by enzyme-linked immunosorbent assay (ELISA). Firstly, 0.5 µg/well of PPD, HspX, and ESAT6 were separately added into the plate at 4°C overnight. Secondly, the plates were blocked with 5% skimmed milk powder, then incubated with the double-diluted serum at 37°C for an hour. And then the plates were washed and added 100 µL of goat anti-mouse IgG (Solarbio, Beijing, China) and rabbit anti-mouse IgG1 and IgG2c (Rockland Immunochemicals Inc., Montgomery, PA, USA) The 3,3′,5,5′-tetramethylbenzidine (TMB) substrate was added at 200 µL/well and incubated at room temperature for 5 min. The reaction was then stopped by diluted sulfuric acid (1 mol/L) at 50 µL/well. The color was quantified at 450 nm. The serum in the PBS group was used as the negative control. The antibody titer was evaluated as a reciprocal of each endpoint dilution.

Total HUVEC protein was extracted using RIPA Lysis Buffer containing protease and phosphatase inhibitors. Protein concentration was measured using the BCA protein assay kit (Solarbio, Beijing, China). Total proteins were separated in 7.5% SDS-polyacrylamide gels and transferred to PVDF membranes (Bio-Rad, Hercules, CA, USA). Primary antibodies and corresponding dilutions were as follows: AKT (1:10000, Abcam, ab179463), p-AKT (1:5000, Abcam, Ab81283), eNOS (1:1000, Abcam, ab199956), p-eNOS (1:1000, Abcam, ab215717), PI3K (1:1000, Abcam, ab191606), p-PI3K (1:1000, #17366, CST). Secondary antibodies and corresponding dilutions were as follows: Goat Anti-Mouse IgG (1:5000, Solarbio, SE131), Goat Anti-rabbit IgG (1:5000, Solarbio, SE134). β-actin (1:1000, Solarbio, K200058M) was used as an internal control. Images are collected using Amersham Imager 600 (GE, CT, USA) and analyzed by ImageJ software. The experiment was repeated three times.

Total proteins were obtained from EC cells through radio-immunoprecipitation assay buffer (R0010; Solarbio). Next, the protein concentration was quantitated by the BCA kit (PC0020; Solarbio). Then, the proteins were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred onto membranes (2215; Millipore, USA). After being blocked with 5% non-fat milk, membranes were reacted with primary antibodies against PCNA (ab92552, 29 kDa, ½,000), E-cadherin (ab231303, 97 kDa, 1 µg/mL), N-cadherin (ab18203, 100 kDa, 1 µg/mL), Vimentin (ab20346, 54 kDa, 1/1,000), VEGFR-1 (ab32152, 151 kDa, 1/2,000), VEGFR2 (ab11939, 151 kDa, 2 µg/mL), epidermal growth factor receptor (EGFR) (ab52894, 175 kDa, 1/2,000), and GAPDH (ab181602, 36 kDa, 1/10,000). Subsequently, the membranes were incubated with the appropriate secondary antibodies goat anti-rabbit IgG (ab205718, 1/5,000) as well as goat anti-mouse IgG (ab205719, 1/5,000), followed by being exposed to the Enhanced Chemiluminescence Substrate (PE0010; Solarbio) for visualization. All the antibodies were bought from Abcam (USA).

The procedure for using the ICT strip is shown in Fig. 1. The preparation of the colloidal gold solution was described previously [37 (link), 49 (link)]. In brief, 0.01% HAuCl4 solution was heated to 100 °C, and then a trisodium citrate solution was added quickly with continuous vigorous stirring. Then, the colloidal gold solution was continuously boiled until the color changed to wine-red. After cooling, the pH of the solution was adjusted to 7.2 using potassium carbonate. Then, 10 ml of the colloidal gold solution placed into a glass bottle into which 100 μl MAb 3G4 (1 mg/ml) was added. Incubation was carried out for 30 min with gentle stirring. After blocking with BSA, the solution was centrifuged for 30 min at 4 °C. The colorless supernatant was discarded and the pellet was re-dissolved with 1 ml PBST (containing 1% BSA).
The ICT strip contained an absorbent pad, nitrocellulose (NC) membranes, a MAb-gold conjugated, pad and a sample pad. The NC membranes were coated with MAb 2G7 as the test line and with goat anti-mouse IgG (Solarbio, Beijing, China) as the control line.