T8660

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.

Cultured cells were washed with PBS for 5 min, fixed in 4% PFA at room temperature for 30 min, then washed twice with PBS every 10 min. The cells were blocked with 5% BSA and 0.3% TritonX-100 in PBS at room temperature for 2 h, then incubated overnight at 4 °C with primary antibody in 3% BSA and 0.1% TritonX-100 in PBS. After washing 3 times with PBS every 10 min, they were incubated at room temperature with secondary antibody and DAPI in PBS for 2 h, then washed 3 times with PBS every 10 min.
The primary antibodies and dilutions were as follows:
Anti-SOX2 (R&D, AF2018, 1:500)
Anti-PAX6 (DSHB, AB-528427, 1:10)
Anti-TUJ1 (Sigma, T8660, 1:5000)
Anti-KI67 (Abcam, ab15580, 1:800)
Anti-CHD7 (CST, #6505, 1:1000)
Anti-GFP (Abcam, ab6673, 1:400)
Anti-MAP2 (Millipore, MAB3418, 1:1000)
Anti-SMI312 (Biolegend, 837904, 1:1000)
Anti-TBR1 (Abcam, ab31940, 1:1000)
Anti-CTIP2 (Abcam, ab18465, 1:200)
Hoechst (Life-tech/3570, 1:2000)
DAPI (Sigma, D9542, 1:1000)