Fig. 7.

The time course of BAPTA retention in the cultured slices parallels exactly the time course of neuroprotection. The cultures were loaded using ¹⁴C-BAPTA-AM, and the relative quantity of ¹⁴C-BAPTA in the slice tissue was assessed autoradiographically. The chelator was fixed at the different times using the cross-linker EDC (see Materials and Methods).A, Representative ¹⁴C-BAPTA autoradiographs of cultures fixed at the indicated times after loading with¹⁴C-BAPTA-AM. EDC was used for panels i–v.vi illustrates a slice similarly loaded using¹⁴C-BAPTA-AM, but fixed in 4% paraformaldehyde (PFA) rather than with EDC immediately after loading. This fixative fails to maintain the chelator in the tissue (compare with i). Scale bar, 1.2 mm. B, Densitometric quantitation of the time course of BAPTA retention. Data shown are background-subtracted mean density values obtained from at least two ¹⁴C-BAPTA-AM-loaded slices at each time point and at each group. PFA-fixed slices were not distinguishable from background. The ¹⁴C-BAPTA signal was retained in the slices for the first 5 hr and then decreased markedly by 24 hr (compare with protective efficacy, Fig. 6C). The background-corrected maximum black level of the photographic emulsion is also shown (triangles) to emphasize that the complete retention of EDC-fixed slices between 0 and 5 hr is not attributable to a saturation artifact.