Fluoro gel aqueous mounting media

Gelatin was removed from thaw-mounted tissues in 37 °C PBS. The following antibodies were used for immunofluorescence: EpCAM (1:100; BD Biosciences, cat # 552370), Ki67 (1:100; Invitrogen, Waltham, MA, cat # MA5-14520), estrogen receptor alpha (ESR1; 1:100; Santa Cruz Biotechnology, Dallas, TX, cat # A0716), progesterone receptor (PGR; 1:100; Invitrogen, cat # MA5-14505), FOXA2 (1:100; Abcam, Cambridge, MA, cat # ab108422), and GFP (1:1000; Invitrogen, cat # A-11122). Tissues were blocked in PBS buffer (1 × PBS, 1% BSA, 10% normal goat serum, and 0.1% Triton X-100) for 1 h at RT before incubation in primary antibodies; EpCAM, Ki67, and GFP antibodies for 1 h at RT, and ESR1, PGR, and FOXA2 antibodies overnight at 4 °C. Tissues were washed thrice for 10 min each before incubation in appropriate species-specific Alexa Fluor-conjugated secondary antibodies (1:1000) for 40 min in the dark (Alexa Fluor 555, Cell Signaling Technology, Danvers, MA, cat # 4417; Alexa Fluor 568 Invitrogen, cat # A11036). After 2–10 min PBS washes, tissues were counterstained with DAPI (300 nM; BioLegend, San Diego, CA) and cover-slipped using fluoro-gel aqueous mounting media (Electron Microscopy Sciences, Hartfield, PA, cat # 17985–30). Omission of primary antibodies served as a negative control. Fluorescent imaging was performed using a Leica 5500 microscope.