Dab horseradish peroxidase color development kit

The cells transfected with the pBmFlag-BlBRC-Z (1–4) plasmid for 48 h were then lysed with RAPI buffer (Beyotime, China) containing one tablet of complete protease inhibitor (Roche, USA) per 100 ml at 4°C on a rocker for 30 min and then scraped. The recombinant proteins were electrophoresed on a 12% SDS-polyacrylamide gel, and then electrotransferred onto a PVDF membrane. After being washed for 5 min in PBST (pH 7.4), the membrane was blocked with blocking solution (5% skim milk, 17 ml of PBST) overnight at 4°C. Subsequently, the membrane was incubated with anti-Flag mouse monoclonal antibodies which were diluted 1:2000 with blocking solution at 37°C for 2 h. After washing three times with PBST (pH 7.4), the membrane was incubated in horseradish peroxidase (HRP) conjugated goat anti-mouse IgG secondary antibodies diluted 1:2000 with blocking solution at 37°C for 2 h. Finally, the membrane was washed three times with PBST, and the bands were detected by the DAB Horseradish Peroxidase Color Development Kit (Solarbio) according to the manufacturer’s instructions.

The IHC assay was performed as previously described [61 (link)]. Briefly, ovarian paraffin sections (six mm thickness) were rehydrated through graded ethanol series. The tissue antigen was activated in citrate-buffered solution, and endogenous peroxidase was quenched in methanol with 3% hydrogen peroxide for 10 min after permeabilizing with 0.3% Triton X-100. The sections were blocked in 5% bovine serum albumin (BSA) for one hour and incubated with the primary antibody at four degrees Celsius overnight. The secondary antibody and third antibody at room temperature, according to the manual of the SABC-AP (rabbit IgG) Kit (SA1052, Boster, Wuhan, China) were added. After Diaminobenzidine (DAB) staining according to the DAB horseradish peroxidase color development kit (DA1015, Solarbio, Beijing, China), the samples were counterstained with Mayer’s hematoxylin (G1080, Solarbio, Beijing, China) for approximately 30 s, dehydrated and dried, then sealed with neutral resin. Positive signals appeared brown, and the images were acquired using a Nikon microscope (T300, Nikon, Tokyo, Japan).