Dako protein blocking solution

Cells were spun on glass slides, fixed with 4% PFA (Sigma-Aldrich, 20649296018) for 10 min at room temperature and washed three times for 5 min with washing buffer (Dako, Glostrup, Denmark, S3006). Afterwards, cells were permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, T8787) in PBS for 10 min at room temperature (RT) and washed three times for 5 min with washing buffer. DAKO Protein Blocking Solution (Dako, X0909) was added and incubated for 1 h at RT. Cells were subjected to immunofluorescence staining with primary antibodies specific for PGRMC1 (Abcam ab48012), ERα (Abcam ab259427), PHB1 (Abcam ab75766) and PHB2 (Cell signaling 14085S) overnight at 4 °C. Afterwards, the slides were washed three times for 5 min with washing buffer and a respective fluorophore labeled secondary antibody (anti-goat: Invitrogen, A11055; anti-mouse: Invitrogen, 745480; anti-rabbit: Invitrogen, A31573) was added to the samples and incubated for 1 h at RT in the dark. The slides were washed three times for 5 min with washing buffer and incubated with DAPI (Thermo Fisher Scientific, 15733122) for 5 min at RT. Antibody incubation steps were performed in a humidified chamber. The slides were washed with distilled water, mounted with Fluorescent Mounting Medium (Dako, S3023) and dried overnight. The cells were examined by fluorescence microscopy using the Axioplan 2 Imaging fluorescence microscope.