Sc 398898

MEECs were grown to an appropriate density and fixed in 4% (w/v) paraformaldehyde for 15 min, permeabilized with 0.5% Triton-X-100 for 15 min, and blocked with 5% bovine serum albumin for one hour at room temperature. After blocking, cells were labeled with the primary antibodies (1:100, rabbit anti-EpCAM, Proteintech, 21050-1-AP; 1: 100, rabbit anti-Mucin1, Abcam, ab109185; 1:100, mouse anti-P63, Abcam, ab735; 1:100, rabbit anti-CD44, Proteintech, 15675-1-AP; 1:100, mouse anti-ERα, Santa Cruz Biotechnologies, sc-71064; 1:100, mouse anti-PR, Santa Cruz Biotechnologies, sc-398898; 1:100, rabbit anti-Vimentin, Abcam, ab137321), and the secondary antibodies (1:100, fluorescently labeled goat anti-mouse lgG-cy3, BA1031, Boster company, Wuhan, China) according to the manufacture’s protocol. DAPI (0.5 mg/ml, D3571, Thermo Fisher) was used to stain the nucleus. Then, the fluorescence was detected by Leica DM4000B fluorescence microscope.

The DAB staining was performed using a commercial DAB Detection Kit (Maixin biotech company). ALI 3D cultures or tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 4 μm sections. The sections were deparaffinized and hydrated through xylene and graded alcohol series and rinsed for 5 min in tap water. Antigens were retrieved by heating samples in a microwave for 15 min in citric acid buffer. The primary antibodies (1:100, rabbit anti-EpCAM, Proteintech, Chicago, IL, USA, 21050-1-AP; 1: 100, rabbit anti-Mucin1, Abcam, Cambridge, UK, ab109185; 1:100, mouse anti-P63, Abcam, ab735; 1:100, mouse anti-ERα, Santa Cruz Biotechnologies, sc-71064; 1:100, mouse anti-PR, Santa Cruz Biotechnologies, sc-398898; 1:100, rabbit anti-Vimentin, Abcam, ab137321) were incubated, respectively, on the slides at 4 °C overnight, then detected with the reaction-amplified reagent for 20 min, and conjugated with high-sensibility enzyme conjugated lgG polymer. Reactants were visualized with the fresh-prepared DAB chromogenic solutions for 3 to 5 min. Hematoxylin somatic cell staining reagent was used to counter-stain nuclei. All the coverslips were mounted on the glass slides using anti-quenching Fluoroshield™ histology mounting medium (Sigma-Aldrich) and visualized under a fluorescence microscope (BX51TF, Olympus company, Tokyo, Japan) with magnification 40 ×.