tPA binds to aggregates proteins formed by injured neurons. (A) Neuronal cultures were perfused with 40 nM tPA⁶³³ and 0.01 g/L TRR and sequential confocal images taken. This experiment was reproduced across 3 independent cultures. (B) Staurosporine (200 nM final) was added to neuronal cultures at various time points after which the conditioned media were replaced with PBS containing 20 μM Congo red and fluorescence was determined by plate reader (λ_excitation = 544, λ_emission = 590). Graph depicts the relative change in TRR fluorescence across 3 independent experiments. Data represent average ± SEM. *P < .05 by 1-way ANOVA with Neumann-Keuls correction. (C) Neuronal cultures were treated with 200 μM glutamate or 200 nM staurosporine for 24 hours (or incubated at 56°C for 0.5 hours as a positive control for cell death and protein aggregation). Cultures were then lysed in PBS containing 1% Triton-X 100. Aggregated proteins are well known to be detergent insoluble.²⁸  The detergent-insoluble fraction was isolated from these lysates by centrifugation (16 000g, 20 minutes), solubilized in SDS-loading buffer with dithiothreitol, and subjected to 10% SDS-PAGE/Coomassie staining. Densitometry of the Coomassie-stained 40- to 80-kDa proteins demonstrates that aggregated proteins increase following injury in neuronal culture: 270% ± 10% for glutamate injury, 250% ± 10% for staurosporine injury, and 320% ± 20% for heat injury relative to uninjured “con” cultures. Data represent average ± SEM from 2 independent experiments performed in duplicate.