Mouse anti p63

Paraffin embedded sections were deparaffinised, rehydrated in an ethanol series, permeabilised in 0.5% Triton-X/PBS and boiled in 10 mM sodium citrate for 11 mins in a pressure cooker for antigen retrieval. Samples were blocked in blocking buffer (10% normal goat serum (Sigma, G9023) in PBS-0.05% Triton-X (VWR, 28817.295). Primary antibodies were diluted in blocking buffer as follows: rabbit anti-β-catenin 1:1000 (Cell Signalling, 957 S), mouse anti-αSMA 1:100 (Abcam, ab7817), mouse anti-E-cadherin 1:500 (BD biosciences, 610182), mouse anti-Cytokeratin-18 pre-diluted (Progen Biotech 65028), rabbit anti-vimentin 1:100 (Cell signalling, 5741), rabbit anti-Cytokeratin-14 (Abcam, ab53115), mouse anti-p63 1:100 (Abcam, ab735), rabbit anti-cleaved caspase-3 1:800 (Cell signalling, 9664) and the nuclear dye Hoechst 33342 at 5 µg/ml (Thermofisher, H3570). The following secondary antibodies were all purchased from life technologies and were all diluted in blocking buffer 1:500: AlexaFluor 488 goat anti-rabbit (A11008), AlexaFluor 647 goat anti-rabbit (A21245), AlexaFluor 488 goat anti-mouse (A11001), and AlexaFluor 647 goat anti-mouse (A21237).

Freshly dissected mammary glands were fixed in 4% paraformaldehyde and embedded in paraffin, or in optimal cutting temperature (OCT) medium (VWR International) for the transplantation experiments. Then, 4 µm sections were de-waxed and rehydrated through xylene and a gradient of ethanol and subjected to antigen retrieval in boiling citrate buffer for 20 min. The tissue was then permeabilized with 0.3% Triton X-100; non-specific antibody binding was blocked with 5% FBS and 10% BSA, and then sequentially incubated with primary and secondary antibodies. Primary antibodies used: chicken anti-GFP (1:1000; Abcam), rabbit anti-K5 (1:500; Covance), mouse anti-K8 (1:500; Covance), rabbit anti-Ki67 (1:500; Abcam), rabbit anti-PR (1:800; SantaCruz), and mouse anti-ERα (1:500; Dako); mouse anti-p63 (1:500, Abcam) and mouse anti-K14 (1:100, Abcam). Fluorochrome-conjugated secondary antibodies included AlexaFluor 488-conjugated anti-chicken IgG, Cy3-conjugated anti-rabbit IgG, Cy3-conjugated anti-mouse IgG for paraffin sections and Cy5-conjugated anti-rabbit and anti-mouse IgG for frozen sections, to avoid fluorophore overlap with the red tdTomato signal, visible exclusively in frozen sections. All secondary antibodies were used at 1:1000 dilutions and were purchased from Molecular Probes and Jackson ImmunoResearch Laboratories, Inc. Sections were counterstained with DAPI (1mg/mL; Sigma).

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 ×.