Triton x 100

Fixed cells permeabilized with 0.1% Triton X-100 (Research Products International Corp., catalog no. 11036) for 5 min, blocked with freshly prepared 5% BSA (Fisher BioReagents, catalog no. BP 1600-100, CAS no. 9048-46-8) for 25 minutes, treated with 1:100 solution of primary antibody in blocking medium at 4 °C overnight. Next, the cells were rinsed thoroughly and stained with 1:200 solution of secondary antibody in PBS for 2 h followed by staining with Acti-Stain^TM 670 Fluorescent Phalloidin (Cytoskeleton Inc., catalog no. PHDN1). Then the cover slides were mounted on glass slides using ProLong Diamond Antifade Mountant (Invitrogen, catalog no. P36961) and sealed with clear nail polish. Primary antibodies: Anti-FAK (D1) mouse monoclonal IgG₁ antibody (Santa Cruz Biotechnology, catalog no. sc-271126). Secondary antibody: Alexa Fluor^® 488 AffiniPure F(ab’)₂ Fragment Donkey Anti-Mouse IgG (H + L) (Jackson ImmunoResearch Laboratories Inc., code. 715-546-151).

Cells collected from 20 samples (18 patients) were fixed with 2% paraformaldehyde (Electron Microscopy Sciences) for 10 min, permeabilized with 0.4% v/v Triton X-100 (Research Products International Corp) for 7 min, blocked with 5% goat serum (Invitrogen) for 30 min, and labeled for 1 h at RT with DAPI (Life Technologies), anti CD45-phycoerythrin (CD45-PE, Clone HI30, BD Biosciences), and a cocktail of primary antibodies to identify CK positive cells (Pan-CK clone AE1/AE3, eBioscience, clone CK3-6H5, Miltenyi Biotec, and CK clone CAM5.2, BD Biosciences). PSA positive cells were identified using a rabbit anti-PSA polyclonal antibody (Dako) and were visualized using AlexaFluor-647 secondary antibodies (Anti-rabbit IgG, Cell Signaling). For three patient samples, PSA staining was replaced by Vimentin (Vimentin-AF467, clone V9, Abcam) and N-cadherin (NC-AF647, clone EPR1791-4, Abcam) staining. After staining, the cells were imaged (Axio Observer Z1, Zeiss) and manually enumerated using specific classification criteria (Fig. 3a, d). For patient samples, purity is calculated as the number of CTCs that meet the criteria described in Fig. 3a (i.e., DAPI+ with CK and/or PSA positivity or nuclear size >9 μm with nuclear/cytoplasmic (N:C) ratio >0.6 and negative for CD45) divided by the total number of DAPI positive cells counted.