Anti pan cytokeratin

Cells were immunostained with primary antibodies at room temperature for 30 min following fixation and permeabilization with 4% paraformaldehyde (PFA) (Cat No. 09154-85, Nacalai-Tesque, Kyoto, Japan) and 0.15% Triton X-100, (Cat No. 93343-100ML, Sigma) respectively. Primary antibodies used were anti-CD45 (Cat No.304002, 1:100 or 304032, 3:100; BioLegend, San Diego, CA), anti-CD45 conjugated with Brilliant Violet 421 (304032, 3:100; BioLegend), anti-EpCAM (ab7504, 1:100; Abcam), anti-CD133 (130-090-422, 1:20; Miltenyi Biotec), anti-vimentin (ab45939, 1:200; Abcam, Cambridge, UK), anti-pan-cytokeratin (628602, 1:100; BioLegend), anti-cytokeratin 19 (628502, 1:100; BioLegend), and anti-CEA (M707229, 1:50; Dako Corp., Carpenteria, CA). For signal amplification, secondary antibodies (A21422, A21235, A11046, A21245, 1:200; Invitrogen, Carlsbad, CA) or a labeling kit (Z25005, Invitrogen) were used. Nuclear DNA was stained with SYTOX Blue (Molecular Probes, Eugene, OR) to confirm the viability of the CTCs. Cell surface markers were immunostained before fixation.

5000 fibroblasts were seeded per well in a 96 well plate in DMEM/10% FBS overnight. Cells were fixed in 10% neutral formalin buffer for 24 hours, permeabilized in methanol for 10 minutes at -20°C and blocked in PBS containing 3% FBS for 1 hour. Cells were then incubated with the following antibodies (1:100) for 24 hours in blocking buffer: anti-PDGFR-α (Cell Signaling Technology, cat no.5241), anti-FSP1 (Abcam, cat no. ab75550), α-SMA (Abcam cat no. 7817), anti-VEGFR2 (Santa Cruz Biotechnology, cat no.sc-393163) and anti-Pan-Cytokeratin (Biolegend, cat no.628602). Cells were washed in PBS 3 times and incubated with the following secondary antibodies (1:1000): anti-rabbit-biotinylated (Jackson Laboratories, cat no.111-065) to detect PDGFR-α or anti-mouse-biotinylated (Vector Laboratories, cat no.BA-9200) to detect VEGFR2, α-SMA or Pan-Cytokeratin. Biotinylated antibodies were incubated with streptavidin bound to horseradish peroxidase (HRP) (Vector Laboratories, cat no. SK4100) for 30 minutes. FSP1 was detected using secondary anti-rabbit-HRP (1:500, Avantor, cat no. 10150-732). Protein expression was detected through HRP reaction to 3, 3 -diaminobenzidine substrate (Vector Laboratories, cat no.SK4100).