Rabbit anti pr

Sections of paraffin-embedded neutralized buffered formalin fixed tissue were used for Ni-DAB (FoxM1) or DAB (other antibodies) colorimetric immunohistochemistry (IHC), while paraformaldehyde fixed frozen tissue sections or cells grown on coverslips were subjected to immunofluorescence (IF). Antibodies include rabbit anti-FoxM1 (1:1000 for IHC or 1:500 for IF; Santa Cruz, cat# sc-502), rabbit anti-cyclin D3 (1:500; Santa Cruz), rabbit anti-Ki67 (1:500; Thermo), rat anti-BrdU (1:200; Abcam), rat anti-phosphorylated histone H3 (Ser 10) (1: 1000; Millipore), rabbit anti-PR (1:300; Santa Cruz), rabbit anti-ERα (1:300; Santa Cruz), and rabbit anti-cleaved caspase 3 (1:300 for IF; Santa Cruz). Counting of immunostaining-positive cells were determined using the Image J program available at http://imagej.nih.gov/ij (NIH, USA), and the analyses were based on examination of serial sections for at least 5–6 IS samples collected from 3–5 different mice for each group.

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).