Male Rat Kangaroo Kidney Epithelial Cells (PtK2 Line)

The technique of double immunofluorescence was employed to simultaneously label a log phase culture of PtK2 cells with mouse anti-tubulin and rabbit anti-beta-catenin primary antibodies, followed by goat anti-mouse and anti-rabbit secondary antibodies conjugated to Cy3 and Cy2, respectively. Tubulin is the basic component of microtubules and members of the catenin family of peripheral cytosolic proteins bind selectively to the highly conserved cytoplasmic tail domain of the cell-to-cell adhesion cadherin proteins. The culture was counterstained with Hoechst 33258, targeting cell nuclei. Images were recorded in grayscale with a 12-bit digital camera coupled to either a Nikon E-600 or Eclipse 80i microscope equipped with bandpass emission fluorescence filter optical blocks. During the processing stage, individual image channels were pseudocolored with RGB values corresponding to each of the fluorophore emission spectral profiles.