Hrp conjugated secondary anti rabbit igg

Briefly, supernatants of Lipofectamine 2000 alone, eGFP mRNA, or Link N mRNA-transfected SCP1 cells and chondrocytes were collected after 24 h. Protein concentrations were determined with the Lowry assay and 10 μg of cell supernatant was pipette onto the nitrocellulose membrane and allowed to air dry. Membranes were then blocked with 5% BSA in TBST for 1 h, followed by 3 washing steps (10 min each). Then, the membranes were incubated overnight with HAPLN1 (TA325115-Origene, Herford, Germany) primary antibody (dilution 1:1000) at 4 °C, and the next day membranes were washed 3 times (10 min each). Afterwards, the membrane was treated with HRP-conjugated secondary anti-rabbit IgG (Santa cruz, dilution 1:10,000) for 2 h. Membranes were developed upon incubating them with ECL substrate solution for a minute. Chemiluminescent signals were quantified using image J analysis software

Total proteins were extracted and resolved by SDS-PAGE, transferred to nitrocellulose membranes, blocked by 5% skim milk in 1× TBST buffer, and incubated with the primary antibody at 4 °C overnight. The following primary antibodies were used: anti-GAPDH (Cell Signaling Technology, cat#2118 L, 1:5000), anti-AKT (Cell Signaling Technology, cat#4685 s, 1:2000), anti-phospho-AKT (Cell Signaling Technology, cat#4060 s, 1:2000) anti-CREB (Proteintech, cat#12208-1AP, 1:1000), anti-phospho-CREB (Cell Signaling Technology, cat#9198 S, 1:1000), anti-estrogen receptor alpha (Abcam, cat#ab32063, 1:1000). HRP-conjugated secondary anti-rabbit IgG (Santa Cruz, cat#sc-2357, 1:10,000) was next incubated for 1 h at room temperature. Signals were visualized with ECL Reagent (ShareBio, cat#SBWB012) using GelDoc XR (Bio-Rad).

Tissue samples were fixed in 10% formalin and paraffin-embedded for immunohistochemical analysis. After deparaffinization, rehydration, and antigen retrieval, liver tissue sections were incubated with 3% H₂O₂, and followed by serum blocking with 10% goat serum in 5% bovine serum albumin (Sigma, United States). Then, liver tissue sections were incubated with the primary antibody against RSPO1 (1:1000 dilution, R&D Systems, United States), RSPO2 (1:1000 dilution, R&D Systems, United States), and RSPO3 (1:1000 dilution, R&D Systems, United States) at 4°C overnight, followed by HRP-conjugated secondary anti-rabbit IgG (1:2000 dilution, Santa Cruz, United States) antibody for 1 h at room temperature. Immunohistochemical staining was carried out using SABC kit (Boster, China). For quantification of the immunostain, cells were counted in six randomly chosen high-power fields at 400-fold magnification by two experienced researchers in a double-blinded manner. Results were scored by the percentage of RSPOs-stained cells as below: < 10% (score 0), 10%~50% (score 1), 51%~75% (score 2), and 76%~100% (score 3). Score 0 and 1 were considered as the weak expression, whereas score 2 and 3 were the positive expression.