Sc 84347

Formalin-fixed, paraffin-embedded uterine endometria were serially sectioned, dewaxed with xylene and rehydrated through a graded alcohol series, and then endogenous peroxidase activity was blocked using freshly prepared phosphate-buffered saline (PBS) containing 3% hydrogen peroxide for 20 min. Antigen retrieval was conducted by autoclaving the samples at 121 °C for 15 min in the presence of EDTA (pH 9.0), followed by incubation in blocking solution for 30 min. Next, the sections were washed with PBS and incubated with antibodies against KLF12 (1:600; sc-84347, rabbit Polyclonal Antibody, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and Nur77 (1:250; 3960, rabbit Monoclonal Antibody, Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C. Subsequently, the sections were rinsed with PBS and incubated with an HRP-conjugated goat anti-rabbit secondary antibody at 37 °C for 20 min. HRP activity was detected using diaminobenzidine (Invitrogen, Carlsbad, CA, USA), and the sections were counterstained with hematoxylin. Control sections were run concurrently with the experimental sections using nonspecific rabbit IgG, and they were similarly pretreated. Nonspecific staining was not detected in the controls [18 (link)].

Proteins were extracted as described previously [17 (link)]. The protein concentrations were measured by Bradford assay (Bio-Rad, Hercules, CA, USA). Equal amounts (25 μg) of protein were separated on a 10% SDS-polyacrylamide gel and transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). Immunoblotting was performed by incubating the membranes with primary antibodies against KLF12 (1:2000; sc-84347, rabbit Polyclonal Antibody, Santa Cruz Biotechnology, Santa Cruz, CA, USA), Nur77 (1:1000; 3960, rabbit Monoclonal Antibody, Cell Signaling Technology, Danvers, MA, USA) and GAPDH (1:10000; AP0063, GAPDH polyclonal antibody, Bioworld Technology, MN, USA), followed by incubation with a goat anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody (1:10000; BS13278, Bioworld Technology, St. Louis Park, MN, USA) and Flag-HRP (1:5000; A8592, Sigma, St. Louis, MO, USA). Detection was performed using an enhanced chemiluminescence kit (Amersham Biosciences Corp., Piscataway, NJ, USA), and densitometric analysis of each band was performed with Quantity-one (Bio-Rad, Hercules, CA, USA) software.

After the endometrial samples were dewaxed, endogenous peroxidase activity was blocked using freshly prepared PBS containing 0.3% hydrogen peroxide for 10 min. Antigen retrieval was conducted by autoclaving the samples at 121 °C for 15 min in the presence of EDTA (pH = 9.0), followed by incubation in blocking solution to block endogenous biotin for 30 min. The sections were washed with TBS and then incubated with antibodies against KLF12 (1:600; sc-84347, rabbit polyclonal antibody, Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4 °C in a humidified chamber. Subsequently, the sections were rinsed with PBS and incubated with an HRP-conjugated goat anti-rabbit secondary antibody at 37 °C for 20 min. HRP activity was detected using diaminobenzidine (Invitrogen, Carlsbad, CA, USA), and the sections were counterstained with hematoxylin. Control sections were run concurrently with the experimental sections using nonspecific rabbit IgG, and these sections were similarly pretreated. Nonspecific staining was not detected in the controls.