tPA binds to injured neurons in vitro. (A) Neuronal cultures were exposed to OGD, glutamate, or staurosporine for 72 hours. Cultures were then perfused with 10 μg/mL 7AAD and 10 nM tPA⁶³³ for 5 minutes and then imaged sequentially by confocal microscopy. Micrographs in the top row represent an overlay of 7AAD fluorescence (red) and tPA⁶³³ fluorescence (white). The bottom row of micrographs shows the corresponding bright-field images. Arrowheads indicate examples of necrotic cells. Arrows indicate examples of apoptotic cells. Appropriate “bleed-through” controls were conducted to ensure specific signal detection (not shown). (B-E) Neuronal cultures were treated with staurosporine (B,D) or glutamate (C,E). At increasing time intervals, cultures were incubated with 400 nM tPA for 15 minutes and washed, and then cellular proteins harvested and subjected to immunoblot analysis. (B-C) Representative immunoblots for tPA, full-length PARP, cleaved PARP (cl PARP), and GAPDH. A Coomassie stain of each membrane is shown as a loading control. “72con” refers to lysates from uninjured cultures at 72 hours. Note, endogenous tPA expression is low in our neuronal cultures and could not be detected by immunoblot analysis (not shown). (D-E) Densitometric quantitations of tPA-binding from 3 independent experiments (normalized for GAPDH levels). Data represent average ± SEM. *P < .05 by 1-way ANOVA with Neumann-Keuls correction.