Immunofluorescent Staining and Quantitative Immunofluorescence Analysis of Apoptotic Neuronal Cells

Immunofluorescence staining was carried out in animals using procedures reported previously ²⁶, ³² with some modifications. In brief, the removed brain tissue was fixed in 4% formaldehyde for 18 h at 4°C and cryoprotected in 30% sucrose solution in PBS. Frozen transverse sections (30 μm) at the level of hippocampus were cut on a cryostat and collected in 0.1 M PBS. Free‐floating sections of the hippocampus were processed for immunoreactivity for NeuN (Chemicon); p‐Drp1(Ser616) (rabbit polyclonal, Novus Biologicals) and activated caspase‐3 (Cell Signaling), as well as a mitochondrial marker, a mouse monoclonal antiserum against ATPB (Anti‐ATPB antibody, Abcam, Cambridge, MA, USA); sections were also stained with 4′,6‐diamidino‐2‐phenylindole (DAPI, Sigma‐Aldrich, St. Louis, MO, USA). Sections were viewed under an Olympus AX‐51 epifluorescence microscope (Olympus, Kyoto, Japan).

Quantitative immunofluorescence analysis of apoptotic neurons in the hippocampal CA3 was performed on representative images from three separate rounds of staining. CellScan (Olympus) was used to assess the images and quantify the relative fluorescent intensity. Each analysis was calculated within a single field (250 μm × 250 μm) in the hippocampus CA3. The numbers of apoptotic cells, defined as those containing condensed chromatin, were manually counted.

Some animals were processed for histopathological analysis of the severity of neuronal death in the hippocampus. For this analysis, 4‐μm paraffin‐embedded brain sections were deparaffinized and stained with cresyl violet.