Immunofluorescence
HEK293 cells were seeded into 2 cm x 1 cm chamber slides at a density of
50,000 cells/slide 24 h prior to transfection. Cells were grown until
they reached 80% confluency. Medium was then removed, and cells washed
3x in PBS before being fixed in 1 mL of 4% (v/v) paraformaldehyde in
PBS for 15 min. Cells were then washed 3x with PBS before being
permeabilized with 1 mL of freshly made 0.05% (v/v) Triton X-100 in PBS
for 5 min. Cells were washed 3x with PBS and blocked for 1 h in 10%
(v/v) normal donkey serum. Cells were washed 3x with PBS before 400 µL
of PBS containing 2% (v/v) normal donkey serum and 1/200 dilutions of
indicated primary antibodies was added. Cells were left in primary
antibody at 4°C overnight. Primary antibodies were removed, and cells
washed 3x in PBS before secondary antibody solution of 400 µL PBS
containing 2% (v/v) normal donkey serum and 1/200 dilutions of donkey
anti-rabbit AF488, AF594 and AF647 were added to each sample. Cells were
incubated under foil at room temperature for 1 h before secondary
antibodies were removed and cells washed 3x with PBS. Cells were mounted
with 400 µL of VectaShield Hardset containing DAPI and cover slips
carefully applied. Cells were incubated overnight in a 5% (v/v)
CO2 incubator before being sealed with nail polish.
Images were collected using a Leica TCS SP8X/MP (Leica Microsystems,
NSW, Australia) confocal microscope and processed with ImageJ software
package.