IF Staining for a single marker: NR2F1, SOX9, or MenaINV

Primary tumor or lung paraffin-embedded sections (4 μm) were first melted at 60 C for 1 h, deparaffinized in xylene, and rehydrated in a graded series of ethanol solutions. Antigen unmasking was performed 1 mM EDTA (pH 8.0) or 1x citrate buffer (pH 6.0) (Diagnostic BioSystems, cat #99990-096) at 97 C for 20 min in a conventional steamer. Slides were rinsed with PBS, permeabilized with 0.5% Triton X-100 in PBS for 5 min at room temperature, and incubated with the blocking buffer solution (10% FBS, 1% BSA, 0.0025% fish skin gelatin in PBS) for 1 h at room temperature. Slides were then incubated overnight at 4 C with primary antibodies against chicken anti-GFP (Novus, cat #NB100-1614, concentration 100 μg/mL) and rabbit anti-NR2F1 (Abcam, cat #ab181137, concentration 5 μg/mL), rabbit anti-SOX9 (Millipore, cat #AB5535, concentration 1 μg/mL), chicken-Mena^INV (generated in the Condeelis Laboratory, concentration 0.25 µg/mL), or rat-Endomucin (Santa Cruz, cat #sc-65495, concentration 2 μg/mL). For the Mena^INV staining, goat anti-GFP (Novus, cat #NB100-1770, concentration 10 μg/mL) was used. Slides were washed three times in PBS containing 0.05% Tween-20 and incubated with a secondary fluorescent antibody (all Invitrogen, concentration 1 μg/mL) for 1 h in the dark at room temperature. After washing, slides were incubated with spectral DAPI for 5 min and mounted with ProLong Gold antifade reagent (Life Technologies, cat #{"type":"entrez-protein","attrs":{"text":"P36980","term_id":"543983","term_text":"P36980"}}P36980). For negative controls, slides were incubated with PBS solution instead of primary antibodies. Slides were imaged on a Pannoramic 250 Flash II digital whole slide scanner (3DHistech) using a 20× 0.75NA objective lens to capture low magnification fields of view.

For quantification of fluorescence signals, high-resolution images were captured using an epi-fluorescence microscope (GE, DeltaVision) with a 100x objective, and CoolSNAP HQ2 CCD camera. Total cell numbers per high-power field (65 × 65 µm², see legend) were counted and the percentages of positive or negative cells were calculated. NR2F1, Mena^INV, and Endomucin channels were thresholded to just above background based upon the negative control.