Cleaved Caspase-3 Assay.

Cortical neurons from wt p75^NTR and p75^NTR KO mice seeded on coverslips were left untreated or transduced with wt/mut p75^NTR. On day in vitro 3 (DIV3), neurons were treated for 12 h with 20 ng/mL human proBDNF. Samples were then fixed in cold 1:1 acetone/methanol solution for 15 min at −20 °C and processed for immunofluorescence with anti–cleaved caspase-3 (1:300, 9664; Cell Signaling Technology) and anti–MAP-2 (1:2,500, M9942; Sigma–Aldrich) antibodies. Samples were imaged on a confocal microscope with a 20× air objective (numerical aperture = 0.5) and pinhole at 1.5 airy units. Cleaved caspase-3–positive neurons were defined as MAP2-positive cells displaying a mean intensity above an intensity threshold in the cleaved caspase-3 channel.