Immunofluorescence Analysis of TPX2 in Cancer Cells

Immunofluorescence analysis was done on the cultured cells according to the protocol described by Wittmann et al. [15] (link). Briefly, M14 and ARO cells were grown on glass coverslips, washed twice with phosphate-buffered saline (PBS) and fixed with cold methanol for 30 minutes. Subsequently, the cells were permeabilized with PBS containing 0,2% TritonX-100 (PBST) for 30 minutes and blocked with PBST containing 2% BSA (Serva). Immunostaining was performed using a TPX2 (D2R5C) XP monoclonal antibody (Cell Signaling Technology) (1∶500 diluted in PBST containing 1% BSA) for 1 hours. Following the washes with PBS, the cells were incubated for 1 h in the dark with a secondary antibody FITC-conjugated anti-rabbit IgG (Jackson Immunoresearch, West Grove, PA) (1∶200 diluted in PBST containing 1% BSA). The nuclei were finally counterstained with diamidinophenylindole 50 ng/ml for 1 minute. After washing with PBS, glass microscopy slides, mounted with Vectashield, were examined by microscope (Leica AF6000 Modular Systems) with 63× oil immersion objective.