Immunofluorescence Staining of Retinoblastoma Cells

Retinoblastoma cells grown in suspension were plated onto poly-D-lysine coated 12-well slides just prior to fixation [48 (link)]. Retinoblastoma cells and R28 cells were fixed with 4% PFA and permeabilized using 0.02% Triton/TBS as described [48 (link)]. Alternatively, cells were fixed and permeabilized with ice-cold methanol followed by acetone at 4°C. Cells were then blocked with 5% NGS in 0.1% BSA/TBS-Tween and incubated overnight at 4°C with primary antibodies in incubating solution containing 1% NGS in 0.1% BSA/PBS-Tween. The antibodies against the following were used: IMPDH1 (Proteintech Cat#22092-1-AP; an antibody raised against the entire IMPDH1 fusion protein), IMPDH2 (Abcam Cat#ab75790), alpha-tubulin (Sigma-Aldrich Cat#T6199), beta-III tubulin (Millipore Cat#MAB1637 and Sigma-Aldrich Cat#T8660), autoantibody It2006 (a kind gift from Edward K. L. Chan), lamin B1 (Abcam Cat#ab16048), detyrosinated tubulin (Millipore Cat#AB3201) and MAP-2 (Abcam Cat#ab32454). Cells were then washed with 0.1% BSA/PBS-Tween and incubated for one hour at 37°C with Alexa fluor® secondary antibodies. Cells were washed with 0.1% BSA/PBS-Tween and mounted with Vectashield Mounting Medium containing DAPI (Vector Laboratories Cat#H-1200) for nuclear staining.