Anti epcam fitc

Antibodies used for western blot, immunofluorescence and flow cytometry were as follows: anti-CD26 (≠AF1180, R&D Systems), anti-CD26 (≠H00001803-D1 Novus Biologicals), anti-E-cadherin (≠610181, BD Biosciences), anti-vimentin (≠MA5-11883, Thermo Fisher Scientific Pierce), anti-EpCAM (≠2929, Cell Signaling Technology), anti-LGR5 (≠TA503316, OriGene Technologies), anti-CD133 (≠MAB4399, Millipore), anti-CD44-FITC (≠44F2, Immunostep), anti-CD133-APC (≠AC133 Miltenyi Biotech), anti-EpCAM-FITC (≠130-098-113, Miltenyi Biotech), anti-LGR5-PE (≠1030-100848, Miltenyi Biotech), anti-CD26-PE (≠26PE, Immunostep), anti-CD26-FITC (≠26F, Immunostep) and anti-E-cadherin-PerCP-Cy5.5 (≠563573, BD Biosciences).
Secondary antibodies used for western blot were horseradish peroxide (HRP)-conjugated antibodies (Sigma-Aldrich). Secondary antibodies used for immunofluorescence were goat anti-rabbit AlexaFluor^®488 and goat anti-mouse AlexaFluor^®594 (Thermo Fisher Scientific).

4T1-GFP cell line was orthotopically injected into the mammary fat pad of 8 week old BALB/c female mice (1 × 10⁵ cells in 50 µl PBS). After 4 weeks, the mice were sacrificed and the tumors were harvested and dissociated in gentle MACS dissociator with enzymatic solution containing 3 mg/ml collagenase (Sigma Aldrich, 11088793001) and 0.1 mg/ml Dnase in RPMI 1640 using the standard program for solid tumors. To receive single cell suspension, the digested cell suspension was filtered through 70 µ strainer with ice-cold PBS. The cells were pelleted and lysed in red blood cell lysis buffer and depleted of CD45+ and EpCAM+ cells as described above. Cells were stained for anti-CD45 FITC (Miltenyi 130-110-658), anti-CD31 FITC (Miltenyi 130-123-675), and anti-EPCAM FITC (Miltenyi 130-117-752), anti-PDPN APC (Biolegend 127410) and anti-Ly6C Pacific Blue (Biolegend 128014). See Supplementary Table 3 for antibodies information. Dead cells were excluded using PI Staining (Sigma Aldrich, P4170). CAF were gated based on PI^-, CD45^-, CD31^-, EpCAM^-, PDPN⁺. For sorting the CAF subpopulation, we used LY6C⁺ as a marker for iCAF and LY6C^- as a marker for myCAF. The cells were sorted using a FACSAria Fusion (BD Biosciences) into FACS tubes containing 1 ml complete DMEM media. The maximum tumor volume of 1000 (mm)³ was not reached in any experiment.