Horseradish peroxidase linked anti mouse or anti rabbit igg

At the end of each experiment, cell culture media was removed, and the cells were washed with ice cold PBS and then lysed with radioimmunoprecipitation (RIPA) buffer containing the Complete Mini^® protease inhibitor cocktail (Millipore Sigma). Total protein content for each sample was quantified using the Bradford protein assay. An equal amount of protein (10 μg) were separated on a 4–12% Bis –Tris gels (Thermo Fisher Scientific) at 125 V for 60 min and transferred onto a polyvinylidene fluoride (PVDF) membrane. The PVDF membranes were blocked with 5% milk in tris-buffered saline with 0.01% Tween for 1 h and then were incubated with polyclonal rabbit anti-human PGRMC1 (1:2000, catalog No. HPA08277, Millipore Sigma), polyclonal rabbit anti-human GR (1:1000 catalog No. 3660S, Cell Signaling) or monoclonal rabbit anti-human B2M (1:10,000, Catalog No. 12851S, Cell Signaling) antibodies overnight at 4°C. The membranes were then incubated with the appropriate secondary antibody (horseradish peroxidase-linked anti-rabbit or anti-mouse IgG at 1:1000 dilution, Cell Signaling) for 1 h at room temperature, after which they were incubated with the SuperSignal^® West Pico Chemiluminescent Substrate (Thermo Fisher Scientific) and exposed on X-ray films. The band densities were quantified using ImageJ^® and both PGRMC1 and GR were normalized to B2M.

The sections were immersed in 0.3% H₂O₂ for 30 min to block endogenous peroxidase. Following, the sections were blocked with normal goat serum (abs933, Absin, Shanghai, China) for 30 min at 25 °C, then incubated with primary rabbit anti-doublecortin (DCX, 1:100 dilution; ab18723, Abcam, Cambridge, UK), mouse anti-neuron-specific nuclear protein (NeuN, 1:100 dilution; MAB377, Millipore Corp, Billerica, MA, USA) or Ionized calcium-binding adaptor molecule 1 (Iba-1, 1:100 dilution; ab153696, Abcam, Cambridge, UK) antibodies overnight at 4 °C. The sections were incubated with horseradish peroxidase-linked anti-rabbit or anti-mouse IgG (1:500 dilution; Cell Signaling Technology, Inc., Beverly, MA, USA) for 60 min at 25 °C, and visualized with DAB substrate kit (Cell Signaling Technology, Inc., Beverly, MA, USA). After washed by PBS, sections were counterstained with hematoxylin, then conducted dehydration and hyalinization. The number of positive cells was observed and assessed under an optical microscope. The ratio was calculated using Image-Pro Plus 6.0 software.

The brain sections were incubated with 0.3% H₂O₂ to eliminate endogenous peroxidase, blocked with 10% goat serum and treated with 0.1% triton X-100 in PBS for 30 min at 25 °C. Then, the sections were incubated with rabbit anti-DCX, mouse anti-NeuN antibodies overnight at 4 °C. After washing with PBS, the sections were incubated with horseradish peroxidase-linked anti-rabbit or anti-mouse IgG (1:500, Cell Signalling Technology, VT, USA) for 60 min at 25 °C and stained with DAB (Cell Signalling Technology, VT, USA). The sections were rinsed with PBS, counterstained with haematoxylin, then dehydrated and observed using an optical microscope.