Validation of ICA results by conventional immunofluorescence. PIR subcellular localization pattern was confirmed by conventional Immunofluorescence staining of melanoma cell lines with anti-PIR antibody detected with goat anti-rabbit Cy3. Left panels show PIR staining, right panels show DAPI (63× oil objective magnification). (a) IGR39 (primary melanoma, nuclear staining); (b) WM115 (primary melanoma, nuclear staining); (c) CaCi1962 (metastasis, mixed staining); (d) AdMa1935 (metastasis, cytoplasmic staining); (e) AnSe1965 (metastasis, cytoplasmic staining). Insets show corresponding confocal images of representative cells (100× oil objective). Scale bar, 10 μm. Original greyscale images were presented to avoid digital pseudocoloring with image editing softwares. PIR and DAPI images were kept separate due to overlapping localization in some samples, and for correct visualization of PIR intensity in case of mixed localization in nuclei and cytoplasm.
Licciulli et al. BMC Cell Biology 2010 11:5 doi:10.1186/1471-2121-11-5