4.3. Centrosome Targeting Sequence Mapping Using Immunofluorescence Staining and Microscopy

U2OS cells were plated onto coverslips in 6-well plates and grown until 60%–70% confluency before transfection of GFP-tagged APC fragment sequences for 48 h (see Section 4.2). Cells were fixed with methanol:acetone (1:1) for 3 min, followed by three washes using PBS. Cells were blocked with 3% bovine serum albumin and stained with primary antibodies. To detect the centrosome, polyclonal PCNT (Abcam ab4448, 1:1500) and PCNT monoclonal (Abcam ab28144, 1:1500) were used. γ-tubulin (Sigma T5192, 1:800) and monoclonal γ-tubulin (Sigma T5326, 1:800) antibodies have also been used to yield a similar localization result. Cells were washed three times using PBS and subsequently incubated with the fluorescence secondary probes Alexafluor-594 (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). Cells were washed extensively before they were mounted with Vectashield (Vector Laboratories Inc., Burlingame, CA, USA). Cells were stained in the dark to prevent bleaching of GFP. Cells were visualized using Olympus BX-51 inverted microscope (Olympus) and optical sections were taken using an Olympus FV1000 confocal microscope (Olympus) at 60× magnification.