Rabbit anti vimentin polyclonal antibody

Cisplatin [cis-diammineplatinum(II) dichloride] was purchased from Sigma-Aldrich, LLC (St. Louis, MO). Quick coating solution used to coat the flask for human neonatal dermal fibroblast (HNDF) cells was purchased from Angio-Proteomie (Shrewsbury, MA). Monoclonal mouse anti-vimentin was purchased from Dako, Inc. (Carpinteria, CA) and rabbit anti-vimentin polyclonal antibody was purchased from Bioss, Inc. (Woburn, MA).

The 3D TTA comprised of A549-RFP cells, HNDF-GFP and unlabeled HPAEC endothelial cell line with or without the unlabeled human NSCLC stem cells were imaged at 10× and 20× magnification using a FV1000 laser scanning confocal fluorescence microscope. The presence of tdTomato and GFP in the TTA were imaged by excitation with argon laser at 532 and 453 nm, respectively. The bright field images taken at 10× were used to compare the size of the TTA in different environmental conditions and cell composition. Images were processed using FV10-ASW 1.7 viewer software (Olympus Imaging America Inc., Center Valley, PA). Captured images were analyzed for sizing using Olympus image analyses software and ImageJ, v. 1.47.
For immunoprobing with vimentin the intact TTA were fixed in 4% paraformaldehyde and washed with phosphate buffered saline (PBS). Blocking was performed in 5% BSA for 30 min, followed by incubation in 1:450 dilution of rabbit anti-vimentin polyclonal antibody (Bioss, Inc.) for 1 h and then later in Alexa Fluor® 633-labeled donkey-anti rabbit secondary antibody at a dilution of 1:500 for 30 min. The TTA were transferred to glass bottom plates for acquisition of confocal images at 10× and 20× magnification using a FV1000 laser scanning confocal microscope (Olympus Imaging America).