Immunofluorescence Analysis of TDP-43 Pathology

iGFP-NLSm were fixed with 4% paraformaldehyde (PFA) in PBS and permeabilized with 0.1% Triton X-100 for 15 min. When indicated, to remove cytoplasmic soluble proteins and quantify the formation of TDP-43 pathology, cells were fixed in 4% PFA containing 1% Triton X-100 for 15 min at room temperature as previously described^{33 (link),34 (link)}. Briefly, after blocking, cells were incubated with the monoclonal antibody phospho-specific p409–410 antibody overnight at 4 °C. After three washes with dPBS, cells were incubated with appropriate secondary antibodies in a blocking buffer for 1 h at room temperature in the dark. Coverslips were mounted with Fluoromount G containing DAPI (Thermo Scientific).