Fig. 6. Caspase-3 immunocytochemistry (A, B), TUNEL (C, E), and bisbenzimide staining (D, F) in E14.5 wild-type (A, C, D) and caspase-3 KO (B, E, F) lumbar spinal cord. A, Immunocytochemistry shows the staining of a cleaved (activated) caspase-3 subunit in the spinal motor neuron column and spinal ganglia in E14.5 wild-type mouse spinal cord (arrowheads), indicating the activation of caspase-3. B, In contrast, no staining of activated caspase-3 subunit is found in E14.5 caspase-3-deficient spinal cord. However, when the TUNEL method is used to detect cell death (C, E), it reveals positive staining in both wild-type (C) and caspase-3-deficient (E) spinal cord, although the staining patterns are distinct. The TUNEL-positive profiles in C andE represent the nucleus of an individual neuron. In wild-type spinal cord, there are more TUNEL-positive nuclei showing punctuate nuclear staining (C), which is correlated with condensed chromatin as revealed by bisbenzimide staining (D). In contrast, there are fewer TUNEL-positive cells in caspase-3-deficient spinal cord (see Results). Moreover, these cells typically show a distinct kind of TUNEL staining (E) and no clear evidence of chromatin condensation as indicated by the size of TUNEL-positive nuclei and reduced bisbenzimide-stained chromatin (F). Although not obvious in this photomicrograph, the nucleus indicated by the arrow inF is weakly labeled with bisbenzimide, indicating little, if any, chromatin condensation. Scale bars: A, B, 400 μm; C–F, 20 μm.