Nestin and Steroid Receptor Figure 3. GR colocalizes with nestin and vimentin in embryonic mouse fibroblasts. Fibroblasts were explanted from Balb/C embryos (E 14.5). (A1–A3) Coincident labelling of cytoplasmic GR and Vim is confined to a subpopulation of cells. (B1–B3) Cytoplasmic accumulation of GR identifies Nes expressing cells. (C1–C3) Incubation with the GR ligand dexamethasone (100 nM) abolishes co-staining of GR with Nes to the benefit of a nuclear GR staining. Bar in Fig. 3, A–C = 5 mm. doi:10.1371/journal.pone.0006084.g003 shortened vimentin filaments. High resolution confocal images showed that the GR label did not evenly decorate the vimentin filaments but exhibited a spotty distribution (Figure 7, A3). A similar staining pattern was obtained in double labeling of nestin and GR (Figure 7C). Quantitative colocalization analysis of confocal microscopy data revealed a high degree of colocalization between GR and tailless vimentin, respectively GR and nestin. We performed both, an intensity correlation coefficient-based analysis and an object based approach. The statistical significance test based on 200 image randomizations [35] showed a significant true colocalization with a P = 100% probability for GR and tailless vimentin respectively GR and nestin. The calculated Pearson’s correlation coefficients were r = 0,825 for GR and tailless vimentin, respectively r = 0,757 for GR and nestin. The distance based analysis [36] revealed 89% positive thresholded pixels in the GR channel and 73% positive thresholded pixels in the vimentin channel. The calculated results for GR/Nes-colocalization were 91% positive thresholded pixels in the GR channel and 84% positive thresholded pixels in the Nestin channel. The data verified that GR was not randomly distributed but colocalized with the filaments composed of tailless vimentin and nestin. PLoS ONE | www.plosone.org 5 June 2009 | Volume 4 | Issue 6 | e6084