Anti rabbit alexa 488 labeled secondary antibodies

Cortical neurons from rats were isolated as described^{50 (link)} and plated onto 12-mm glass coverslips coated with laminin (BD Biosciences) and poly-D-lysine (Millipore), then transfected at 4 days in vitro with pGW1-mApple and pGW1-TDP43(WT)-EGFP or pGW1-TDP43(A315T)-EGFP. Immunocytochemistry was performed as described^{50 (link)} using rabbit anti-TDP43 polyclonal antibodies (Proteintech; 1:1000) and anti-rabbit Cy5-labeled secondary antibodies (Jackson Immunochemical; 1:250). For LC3 immunocytochemistry, neurons were treated for 24 h at 37°C with the indicated compound or DMSO, then fixed and probed with rabbit anti-LC3 primary antibodies (Novus Biologicals, 1:200), and anti-rabbit Alexa 488-labeled secondary antibodies (Invitrogen, 1:500). Nuclei were stained by a brief incubation in PBS supplemented with Hoechst dye (10 μM final concentration). Immunocytochemical samples were imaged using a Zeiss LSM510 confocal microscope equipped with Meta. The total normalized TDP43 level was determined by comparing the intensity of TDP43 immunolabeling in transfected cells to that in non-transfected cells within the same high-powered (63×) field.