IHC Analysis

Sections were deparaffinized in xylene, rehydrated through graded alcohols, and washed in phosphate buffered saline. For p16^Ink4a, antigen retrieval was achieved by incubating in pepsin solution (catalog number AP9007005; Thermo Fisher Scientific) for 5 minutes. The other protein antigens were retrieved by microwaving sections in 10 mmol/L sodium citrate buffer (pH 6.0) for 20 minutes. For BrdU immunohistochemistry, sections were treated with 2N hydrogen chloride for 20 minutes. Sections were treated with blocking buffer for 1 hour and then with a primary antibody overnight. Detailed antibody information and immunohistochemistry conditions are described in Table 1. After extensive washing in phosphate buffered saline, sections were incubated with Alexa Fluor 488–conjugated anti-rabbit IgG (catalog number A11008; Life Technologies, Carlsbad, CA) or anti-mouse IgG (catalog number A11001; Life Technologies). Sections were then incubated in Hoechst 33258 solution (10 μg/mL; catalog number B2883; Sigma-Aldrich) for 30 seconds to stain nuclei. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed by using an ApopTaq Fluorescein in situ apoptosis detection kit according to the manufacturer's instructions (catalog number S7110; EMD Millipore, Burlington, MA). Slides were mounted using gelvatol mounting medium.

Antibodies and Conditions for IHC

BrdU, bromodeoxyuridine; ER, estrogen receptor; MCM, minichromosome maintenance complex component; PR, progesterone receptor.