Immunocytochemistry
rAAV producing cells were harvested 24 hours after nocodazole addition.
1 x 106 cells were spun down at 250xg for 5 min,
culture media was removed, and cells resuspended in 1 mL PBS.
Resuspended cells were incubated with no agitation for 30 min at RT and
allowed to adhere by gravity sedimentation to lysine-coated glass
coverslips in 24-well plates. After 30 min, PBS and unadhered cells were
removed by gentle aspiration. Adhered cells were fixed by 10min
incubation at RT with 0.5 mL 4% (v/v) paraformaldehyde in PBS. Fixed
cells were washed with PBS before permeabilization with 0.5 mL 0.5%
(v/v) Triton-X100 for 10 min. Permeabilised cells were blocked by 10%
normal goat serum (Life Technologies, Carlsbad, CA; #016201) in PBS for
30 min. Cells were then incubated with anti-Fibrillarin antibody (Abcam,
Cambridge, UK; ab5821, 0.1 µg/mL in blocking buffer) for 1 h at RT
before incubation with goat anti-Rabbit Alexa-594 conjugated secondary
antibody (Abcam; ab150080, 1:1000 dilution in blocking buffer) for 45
minutes at room temperature. Coverslips were transferred to a glass
slide and mounted with Fluoroshield mounting media containing DAPI
(Sigma-Aldrich; F6507) for visualisation of nuclei. Slides were imaged
using a Nikon Eclipse Ti fluorescent microscope and images adjusted for
brightness and contrast using ImageJ software.