4 6 diamidino 2 phenylindole 2hcl dapi

Example 5

To isolate stromal cells, tumors were removed and washed with PBS (Ca/Mg free), cut into 1 mm pieces and incubated at 37° C. for 30 min in a digestion solution composed of DMEM (Gibco), 0.5 mg/ml collagenase D (Roche), 0.13 U/ml Liberase TL (Roche) and 1 U/ml DNase 1 (Invitrogen). After 30 minutes, the digested fraction of the mix was collected and centrifuged in DMEM 10% FCS. Remaining undigested tissues were subjected to 1 or 2 additional cycles of digestion, collected and pressed through a 100-μm mesh. For FACS staining, cells were first pre-incubated with mAb 2.4G2 to block Fcγ receptors, and then incubated with the indicated Abs for 40 min in a total volume of 100 μl of PBS containing 2 mM EDTA and 2% bovine serum (PBS-F), followed by appropriate secondary Abs for 30 min when necessary, centrifuged in 2 ml PBS-F and dissolved in 200 μl of PBS-F for FACS analysis. Cells were incubated for 1 min with 4′6-diamidino-2-phenylindole-2HCl (DAPI) (Sigma) prior to analysis to exclude dead cells. Cell doublets were systematically excluded during analysis. Cells were analyzed with Fortessa (BD Biosciences) or Cyan ADP (Beckman Coulter), and Flowjo software (Tristar). Cells were sorted with FACS Aria 3 (BD Biosciences) to 95-98% purity.

Example 4

Tissue processing and staining procedures for immunofluorescence have been described previously (Peduto, L., et al., J. Immunol. 182, 5789-5799. (2009)). Briefly, tissues were fixed O/N at 4° C. in 4% paraformaldehyde (PFA) (Sigma), washed O/N in PBS, incubated in a solution of 30% sucrose (Sigma) until the samples sank, embedded in OCT compound 4583 (Sakura Finetek), frozen in a bath of isopentane cooled with liquid nitrogen and stocked at −80° C. Frozen blocs were cut at 8 μm thickness and sections were processed for staining: after blocking with 10% bovine serum in PBS containing 1% Triton (PBS-XG) for 1 hour at room temperature (RT), slides were incubated with primary antibodies (Abs) in PBS-XG overnight at 4° C., washed 3 times 5 min with PBS-XG, incubated with secondary conjugated Abs or streptavidin for 1 hour at RT, washed once, incubated with 4′6-diamidino-2-phenylindole-2HCl (DAPI) (Sigma) 5 min at RT, washed 3 times 5 min and mounted with Fluoromount-G (Southern Biotechnology Associates). Slides were examined with an Axiolmager M1 fluorescence microscope (Zeiss) equipped with a CCD camera and images were processed with AxioVision software (Zeiss). Mosaic images were generated using Spinning Disk Confocal microscopy (Cell Voyager) and images were analysed with ImageJ software.