Goat anti rabbit igg hrp antibody

Western blot analysis was performed as previously reported [26 (link)]. Total protein was prepared with the RIPA lysis reagent (R0010, Solarbio). After quantification with the BCA Protein Assay Kit, protein samples were separated on the SDS-PAGE gel and transferred onto PVDF membranes (IPVH00010, Millipore, USA). Membranes were incubated with primary antibodies, including Nfatc3 antibody (1:1000 dilution; 18222-1-AP, Proteintech), Pou3f1 antibody (1:1000 dilution; A19330, ABclonal), iNOS antibody (1:1000 dilution; A0312, Abclonal), COX-2 antibody (1:1000 dilution; A1253, Abclonal) and GAPDH antibody (1:10000 dilution; 60004-1-Ig, Proteintech). The Goat anti-Rabbit IgG/HRP antibody (1:3000 dilution; SE134, Solarbio) and Goat anti-Mouse IgG/HRP antibody (1:3000 dilution; SE131, Solarbio) were used as secondary antibodies. The protein signals were visualized using the ECL Western Blot Substrate (PE0010, Solarbio).

Lumbar 4–5 spinal cord segments were collected as mentioned above. Tissue samples were homogenized with radioimmunoprecipitation (RIPA) buffer (Thermo Fisher Scientific, MA, USA) plus phosphatase inhibitor cocktail (100×, Thermo Fisher Scientific, MA, USA). After homogenization, tissues were incubated for 15 min on ice and centrifuged at 13,000× g at 4 °C for 20 min. The supernatant was assayed using the Bradford protein assay (BIO-RAD, CA, USA). Thirty micrograms of protein samples were loaded and run on a 8% Tris-glycine sodium dodecyl sulfate-polyacrylamide gel followed by electrophoresis, then were transferred to a nitrocellulose membrane (BIO-RAD, CA, USA). The membranes were blocked with 5% skim milk in 0.05% Tris Buffered Saline with Tween 20 (TBS-T). After blocking, incubated with primary antibody for overnight at 4 °C with rabbit anti-GAD65 (Cell Signaling Technology, MA, USA, 1:1000) in 5% skim milk. After rinsing the membrane with TBS-T, it was incubated with secondary antibody for 2 h at room temperature with Goat anti-rabbit Ig G/ HRP antibody (Solarbio, Beijing, China, 1:2000) in 5% skim milk. Bands were detected using enhanced chemiluminescence (ECL) solution (Donginbio, Seoul, Korea, A:B = 1:1) and imaged with davinch. Density image was quantified by using image J. GAD65 bands were normalized using the amount of β-actin.

Total proteins were extracted from both tumor and adjacent thyroid tissues of PTC and iPTC patients (Table 1). For each sample, 50μg protein was loaded into the wells of SDS-polyacrylamide gels (10%). Gels were run for separation at 80 V and then at 120V. Proteins were transferred onto polyvinylidene fluoride (PVDF) membranes by using the Bio-Rad Mini-Trans-Blot system. The membranes were then blocked with 5% fat-free milk in PBST (PBS and 0.1% Tween-20) for 2h at room temperature, followed by incubation overnight at 4°C with primary antibody dissolved in blocking buffer. The antibodies used were rabbit monoclonal anti-Versican (VCAN) antibody (dilution 1:1000, Abcam, ab177480), rabbit polyclonal anti-Cadherin16 antibody (dilution 1:400, Proteintech, 15107-1-AP), and rabbit-based anti-β tubulin antibody (dilution 1:1000, Cell signaling cycle, #2146). The secondary antibody was goat anti-rabbit IgG/HRP antibody (dilution 1:1000, Solarbio, SE134), dissolved in blocking buffer and incubated for 1 h at room temperature. Images were acquired by Amersham Imager 600 (GE, USA). Gray values were obtained using a fixed size rectangle feature enclosing the band of interest to obtain the intensity output (median intensity of pixels of the rectangle area) after background subtraction and contrast enhancement by Image J program.