Human autopsy tissue was obtained from the University of Pennsylvania Center for Neurodegenerative Disease Research Brain Bank as described (53 (link)). Informed consent from next of kin was obtained for every case.
Isolation was performed exactly as previously described (52 (link)). Briefly, mid-frontal neocortex of all cases were dounce homogenized using pestil B (Kimble Chase) in 0.25M sucrose and adjusted to final molarity of 1.6M sucrose in TKM. The homogenate was spun on a 1.8M sucrose cushion on the Beckman Coulter XPN-80 ultracentrifuge at 40,000g for 40 minutes at 4°C (Beckman Coulter Inc, Indianapolis, IN, USA). Isolated nuclei were stained with Alexa Fluor 647 conjugated to 2089 (rabbit polyclonal C-terminal anti-TDP-43 antibody, Center for Neurodegenerative Disease Research, University of Pennsylvania), Alexa Fluor 488 conjugated NeuN (EMD Millipore, Billerica, MA, USA), and DAPI (Invitrogen, Carlsbad, CA, USA). Alexa Fluor 647 was conjugated to 2089 according to the APEX Alexa Fluor 647 Antibody labeling kit protocol (Thermo Fisher Scientific, Waltham, MA, USA). Stained nuclei were sorted for single cells containing TDP-43 and NeuN on the BD FACSAria II (BD Biosciences, San Jose, CA, USA) at 20 psi on 100μm nozzle.
Isolation was performed exactly as previously described (52 (link)). Briefly, mid-frontal neocortex of all cases were dounce homogenized using pestil B (Kimble Chase) in 0.25M sucrose and adjusted to final molarity of 1.6M sucrose in TKM. The homogenate was spun on a 1.8M sucrose cushion on the Beckman Coulter XPN-80 ultracentrifuge at 40,000g for 40 minutes at 4°C (Beckman Coulter Inc, Indianapolis, IN, USA). Isolated nuclei were stained with Alexa Fluor 647 conjugated to 2089 (rabbit polyclonal C-terminal anti-TDP-43 antibody, Center for Neurodegenerative Disease Research, University of Pennsylvania), Alexa Fluor 488 conjugated NeuN (EMD Millipore, Billerica, MA, USA), and DAPI (Invitrogen, Carlsbad, CA, USA). Alexa Fluor 647 was conjugated to 2089 according to the APEX Alexa Fluor 647 Antibody labeling kit protocol (Thermo Fisher Scientific, Waltham, MA, USA). Stained nuclei were sorted for single cells containing TDP-43 and NeuN on the BD FACSAria II (BD Biosciences, San Jose, CA, USA) at 20 psi on 100μm nozzle.
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