Alex fluor 488 conjugated igg

Cell immunostaining was performed as described previously [73 (link)]. Briefly, cells were fixed with 4% PFA and then blocked with 10% normal goat serum and 0.1% Triton X-100 in PBS. The cells were then incubated with primary antibodies overnight. On the next day, the cells were incubated with the secondary antibodies for 90 min and DPAI for 5 min. The cells were kept in PBS for observation. Images were taken with a Leica DMi8 fluorescence microscope. The following antibodies were used: anti-T mAb (1:100, Abcam, Catalog No. ab209665), anti-Sarcomeric α-Actinin mAb (1:100, Abcam, Catalog No. ab9465), anti-α-SMA mAb (1:100, Santa Cruz, Catalog No. sc32251), anti-Sox1 mAb (1:100, Abcam, Cat No. ab109290), goat anti-rabbit Alex Fluor 488-conjugated IgG (1:500, Invitrogen, Catalog No. A11008), goat anti-mouse Alexa Fluor Plus 555-conjugated IgG (1:500, Invitrogen, Catalog No. A32727).

Cell suspension after TryPLE digestion from culture was fixed with neutral-buffered 2% paraformaldehyde and blocked with 2% bovine serum albumin (BSA) and 2% normal goat serum (NGS, Sigma-Aldrich). The samples were incubated with antibodies against cell surface epitopes (CD14, 31, 73, 90, 105, 166, EGFR, ITGB4, OCLN and ZO1 (Additional file 1: Table S1), followed by AlexFluor488-conjugated IgG (Invitrogen). For the detection of intracellular epitopes (CDH1 and CDH2) (Additional file 1: Table S1), the cell samples were permeabilized with 1% Triton X-100 before blocking and antibody incubation. The staining signal was analyzed by FACSVerse System (BD Biosciences) with a minimum of 10,000 events in each experiment.