Hematoxylin and eosin staining and immunohistochemistry were performed as previously described13 (link),42 (link). For immunohistochemistry, paraformaldehyde-fixed, paraffin-embedded sections were incubated with the following primary antibodies: ESR1 (sc-8005), PGR (sc-538), CEBPB (sc-150), FOXA2 (sc-6554), αSMA (sc-53142, Santa Cruz, Santa Cruz, CA, USA). The sections were stained with the Vectastain ABC Kit (Vector Laboratories, Burlingame, CA, USA). Immunofluorescence analysis was performed with Alexa Fluor protein-conjugated secondary antibodies (Thermo Fisher Scientific, Waltham, MA, USA) and counterstained with Hoechst 33342 (Sigma).
For EdU-immunostaining, mice were injected with EdU at 50 mg/kg body weight. One hour after the injection, animals were euthanized and tissues collected. EdU-incorporated cells were detected using Click-iT EdU Imaging Kits (Themo Fisher Scientific) as described in the manufacture’s protocol. In some samples, ESR1 (sc-8005) was detected with Alexa Fluor protein-conjugated secondary antibodies (Thermo Fisher Scientific) and immunofluorescent imaging. More than 3 animals were analyzed, and representative pictures are shown.
For EdU-immunostaining, mice were injected with EdU at 50 mg/kg body weight. One hour after the injection, animals were euthanized and tissues collected. EdU-incorporated cells were detected using Click-iT EdU Imaging Kits (Themo Fisher Scientific) as described in the manufacture’s protocol. In some samples, ESR1 (sc-8005) was detected with Alexa Fluor protein-conjugated secondary antibodies (Thermo Fisher Scientific) and immunofluorescent imaging. More than 3 animals were analyzed, and representative pictures are shown.