Alexa fluor 564 conjugated goat anti rabbit antibodies

PBMCs, THP-1 cells, or mouse renal tissue was washed with phosphate buffered saline (PBS) and fixed with 4% paraformaldehyde at room temperature for 20 minutes, followed by permeabilization and blocking with PBS containing 0.1% Triton X-100 and 5% FBS for 1 hour. After another wash with PBS, immunostaining was performed by incubating the cells overnight at 4°C with the appropriate primary antibody (diluted 1:100). Primary antibodies were diluted in PBS containing 0.1% Triton X-100, 0.2% BSA, 0.5 mM phenylmethylsulfonyl fluoride, and 1 mM dithiothreitol. After washing with PBS, secondary Alexa Fluor 488—conjugated goat anti-mouse antibodies or Alexa Fluor 564—conjugated goat anti-rabbit antibodies (diluted 1:200; Invitrogen) were added for 2 hours at room temperature. Samples were nuclear counterstained with DAPI, mounted, and visualized with an LSM510 confocal imaging system (Zeiss).

Human APL samples were kindly provided by Dr. Eduardo Rego from Brazil. Tissue samples were washed with PBS and fixed with 4% paraformaldehyde at room temperature for 20 min, followed by permeabilization and blocking with PBS containing 0.1% Triton X-100 and 5% FBS for 1 h. After another wash with PBS, immunostaining was performed by incubating the cells with mouse anti-Pin1 (Home-made, 1:1000), or rabbit anti-PML (H-238, Santa Cruz; 1:100) primary antibodies at 4°C overnight. Primary antibodies were diluted in PBS containing 0.1% Triton X-100, 0.2% BSA, 0.5 mM PMSF and 1 mM dithiothreitol. After washing with PBS, secondary Alexa Fluor 488-conjugated goat anti-mouse antibodies or Alexa Fluor 564-conjugated goat anti-rabbit antibodies (Invitrogen; 1:200) were added at room temperature for 2 h. Samples were nuclear counterstained with 4,6-diamidino-2-phenylindole (DAPI), mounted and visualized with LSM510 confocal imaging system. For centrosome duplication assays, NIH3T3 cells were used as described previously^{7 (link)}. Briefly, cells were synchronized in G1/S phase by adding 10 μg/ml aphidicolin for 24 h, then fixed with 4% paraformaldehyde at room temperature for 20 min, and stained for centrosomes with anti-γ-tubulin antibodies (GTU-88, Sigma; 1:100) and analyzed by confocal microscopy.