Sp5 2 aobs confocal laser scanning microscope

K562 cells grown in suspension culture were collected via centrifugation at 1400 x g for 3 mins. Nuclei were isolated using the Nuclei EZ Prep nuclei isolation kit (Sigma-Aldrich). Nuclei were fixed in 4% (v/v) paraformaldehyde and rotated at room temperature for 15 mins, then incubated with 50mM NH4Cl for 15 mins, rotating, then washed three times in PBS. The nuclei were then incubated in blocking buffer (2% (w/v) BSA, 0.25% (w/v) Gelatin, 0.2% (w/v) Glycine, 0.2% (v/v) Triton X-100 in PBS)
for 1 hour, rotating at room temperature. Primary antibody was diluted in PBS with 1% (w/v) BSA, 0.25% (w/v) gelatin and 0.2% (v/v) Triton X-100 and incubated for 1 hour, rotating at room temperature. Nuclei were washed three times with washing buffer (0.2% (w/v) Gelatin in PBS). Fluorescent secondary antibody was then applied for 45 mins in the same buffer as the primary antibody, and samples were rotated in the dark. Nuclei were washed three times in washing buffer before 10 mins incubation with DAPI solution. Nuclei were resuspended in a minimum volume of DABCO mounting media (Sigma-Aldrich) to mount onto poly-lysine coated slides.
Samples were viewed using a Leica SP5-II AOBS confocal laser scanning microscope attached to a Leica DM I6000 inverted epifluorescence microscope with oil 63x lens. Images were processed using ImageJ or Volocity 6.3 software.