Immunofluorescent Staining of Primary Neurons

After 2–28 days of growth primary neurons were fixed in 4% paraformaldehyde (PFA) in PBS, washed three times x 5 minutes in PBS, incubated over night at 4°C in PBS containing 10% sucrose and then frozen at -20 until used. The cells were then washed in PBS and blocked in blocking buffer (PBS pH 7.2 containing 0.25% BSA and 0.25% Triton-X-100) for 1hr at room temperature. The primary antibodies, anti-ZNF804A (D14, Santa Cruz) diluted 1:50 and doublecortin (Abcam) diluted 1:2000 in blocking buffer, were incubated over night at 4°C. The specificity of the anti-ZNF804A antibody was verified by Western blot of ZNF804A transfected HEK cells, as others have done previously [23 (link)]. Following primary antibody incubation, cultures were washed in PBS containing 0.25% Triton-X-100 and incubated with secondary antibodies (Alexa Fluor 594 anti-goat IgG and Alexa Fluor 488 anti-rabbit both diluted 1:600, Invitrogen) diluted in blocking buffer. Controls were made with only secondary antibody to avoid investigating unspecific labelling of cells. The slides were then washed twice and mounted in DTG mounting media (2.5% DABCO (Sigma-Aldrich), 50 mM Tris-HCl pH 8.0, 90% glycerol) with or without 0.375 mg/ml DAPI (Sigma-Aldrich). Culture slides were analysed at Olympus FV1000 confocal laser scanning microscope. Some cells were analyzed in three dimensions by confocal laser-scanning microscopy.