Irdye 800 conjugated anti mouse igg antibodies

Treated iPSn were immediately fixed in 4% formaldehyde for 15 minutes and washed three times with PBS. To permeabilize the cells, they were incubated with PBS containing 0.3% Triton X-100 for 30 minutes, then blocked with Odyssey blocking buffer (Li-Cor Biosciences) for 1 hour. Anti-neurofilament antibody (1:750, mouse IgG, Biolegend, SMI-312, #837904) was then incubated overnight in blocking buffer at 4°C, followed by washing in PBS with 0.1% Tween for 20 minutes. IRdye 800-conjugated anti-mouse IgG antibodies (1:1000 dilution, Li-Cor Biosciences) was then added in blocking buffer containing 0.2% Tween for 1 hour. The order of the treatment and placement in the cell culture wells was changed in each experiment to account for any potential technical variations.

iPSC-neurons were seeded on a PDL/laminin coated 96 well plate and analyzed and treated with either lentivirus expressing ykt6 CS vs empty vector, or DMSO vs 5nM FTI (LNK-754) for 14 days. Cells were fixed in 4% paraformaldehyde for 20 minutes and incubated with PBS containing 0.3% TritonX-100 for 20 minutes, then blocked with Odyssey blocking buffer (Li-cor) for 1 hour. Anti-neurofilament antibody (1:1000, mouse IgG 2H3, Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA) was incubated overnight in blocking buffer at 4°C, followed by washing in PBS with 0.1% Tween for 20 minutes. IRdye 800-conjugated anti-mouse IgG antibodies (1:1000 dilution, Li-cor) was incubated in blocking buffer for 1hour, and CellTag™ 700 (Li-cor) was added with the secondary antibody to use for cell normalization. Cells were washed four times in PBS- 0.1% Tween and scanned on an Odyssey infrared imaging system (Li-cor). Neurofilament intensity was determined using Image Studio software (version 2.1 Li-cor) and normalized to cell volume. Replicates represent values from four individual culture wells and were analyzed by Student’s T-test using GraphPad Prism software V 6.0. The sample identities were not blinded.