Fluorochrome tagged secondary antibody

Briefly, MDA-MB-231 cells were seeded on coverslips in 35 mm dishes and treated with siRNA respectively for 24 h, then cells were fixed with 4% paraformaldehyde. Fixed cells were incubated with anti-CD44 (1:100, Cat No. 6124) and CD24 antibodies (1:100, Cat No. 64064) (Abcam, Hangzhou, China) and then the fluorochrome-tagged secondary antibody (1:500, FITC-conjugated anti-rabbit, TRITC-conjugated anti-mouse) (Abcam, Hangzhou, China). Following stained with Hoechst33342 in PBS buffer, coverslips were mounted on slides.

hMSCs after induction with both the induction protocols, were labelled for Nestin (1:100), MAP2 (1:200), TH (1:100), synaptophysin (1:100, ab14692), DAT (1:100), GFAP (1:100, #ab4674), TPH2 (1:150, #ab111828), Ach (1:150, #ab2803) and S100 (1:100, #ab124805) as previously described^{7 (link)}. Same antibodies were used for all the experiments. The dilutions used were titrated before performing final experiments. Briefly, the cells were incubated with primary antibodies for 1 h 20 min at 4 °C, followed by washing and incubation with secondary antibody labelled with fluorochrome-tagged secondary antibody (dilution of 1:400, abcam, USA) for 30 min at RT. The cells were then washed and suspended in PBS and acquired on BD LSR II flow cytometer (Becton Dickinson, USA) with a minimum of 10,000 events for each sample and analysed with FACs DIVA software (version 6.1.2). All the antibodies were procured from Abcam, USA.