tPA and plasminogen bind to injured neurons via different mechanisms. Neuronal cultures were incubated in B27 protein–containing media supplemented with 400 nM tPA, 100 nM plasminogen, 10 mM TXA, or 400 nM reteplase or 800 nM uPA for 15 minutes. Cultures were then washed, fixed, and subjected to anti-tPA/anti-plasminogen/anti-uPA immunofluorescence. Samples were imaged by confocal Z-stack analysis. Shown are maximum projection micrographs where immunopositive signals (white) and nuclear counterstain signals (red) are overlaid. These results were reproduced across at least 3 independent experiments. Note, the immunopositive signals of nonviable cells are so intense that the nuclear counterstain of these cells is not seen in the overlay. In addition, no comparable immunopositive cells were found in sections that were incubated in the absence of tPA, plasminogen, or reteplase. Immunoblot analysis demonstrated that the anti-tPA antibody binds tPA and reteplase with comparable affinities (not shown). Binding to nonviable cells was still apparent when the concentration of tPA was lowered to 50 nM (not shown).