Erα antibody

E12.5, E13.5 and E14.5 mouse embryos were proceeded for paraffin-embedded classical histology and were sectioned into 5-µm thickness. The sections were mounted on positively charged slides. The slides were deparaffinized using Histolemon and re-hydrated. Epitopes retrieval was performed by incubation in 0.05% citraconic anhydride buffer (pH 7.4) at 100  °C for 15 min. Endogenous peroxidase was inhibited by incubation in 30% H₂O₂ for 20 min. Endogenous biotin molecules were blocked with endogenous avidin/biotin blocking kit (ab64212), and non-specific binding was blocked by incubation with 10% goat serum diluted in PBS for 1 h. Subsequently, the sections were incubated overnight in a humid chamber at RT 1 h with ERα antibody (1/200; ab32063 abcam). Sections were extensively washed and incubated for 1 h with the biotinylated anti-rabbit IgG secondary antibody (1/500; ab97049 abcam). Sections were extensively washed and incubated 1 h with HRP-conjugated streptavidin (1/1000; ab7403 abcam). The sections were finally treated with diaminobenzidine in the dark, washed, then rapidly counterstained with Mayer’s Hemalun and mounted in Eukitt medium.

MCF-7R and T47DR cells were fixed in 4% polyoxymethylene at 4°C for 20 min, washed with PBS and permeabilized in 0.1% Triton X-100 at room temperature for 10 min. Cells were then blocked in 10% goat serum at room temperature for 30 min, and incubated with ERα antibody (1:100, Abcam, #16660) in 10% goat serum at 4°C overnight. Cells were washed, incubated with secondary antibodies at room temperature for 1 h, washed, incubated with DAPI at room temperature for 15 min. Slides were then covered with fluorescently quencher 30 μl, sealed and photographed with an Olympus confocal microscope.