Ix71 inverted multicolour fluorescent microscope

Tissues were fixed in Zn-formalin, paraffin embedded and stained as previously described15 (link). In brief, after sections were deparaffinized, rehydrated and subjected to antigen retrieval, sections were blocked in 5% donkey serum for 1 h at room temperature (RT), incubated with primary antibodies for 1 h at RT, washed, incubated with secondary antibodies for 1 h at RT, washed and mounted. Rabbit anti-Zeb1 (Santa Cruz Biotechnology, Santa Cruz, CA) required additional tyramide signalling amplification (PerkinElmer, Waltham, MA). See Supplementary Table 2 for a list of antibodies used. Slides were visualized using an Olympus IX71 inverted multicolour fluorescent microscope.

Female tumour-bearing mice between 12–14 weeks of age were injected with 25 μg kg⁻¹ Texas red-conjugated dextran (70,000 MW; Thermofisher Scientific, Waltham, MA) 30 min prior to euthanasia. Tissues were embedded in OCT (Electron Microscopy Sciences, Hatfield, PA), cut into 5 μm sections and imaged on an Olympus IX71 inverted multicolour fluorescent microscope.