Bx5 fluorescence microscope imaging system

A mitochondrial membrane potential (MMP) assay kit with 5,5′,6,6′-tetrachloro-1,1′,3,3′ tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) [C2006; Beyotime Institute of Biotechnology, Shanghai, China] was used to detect MMP. JC-1 accumulates to form J-aggregates and emits red fluorescence (Cy3, excitation/emission wave length of 525/590 nm) in mitochondria with higher membrane potentials, while JC-1 monomers emit green fluorescence (fluorescein isothiocyanate [FITC], excitation/emission wave length of 490/530 nm) in mitochondria with lower membrane potentials. A decrease of the ratio (red: green) was thus interpreted as a decrease in MMP [33 (link)]. For this assay, different groups of 0.1 mL of purified mitochondria (protein concentration 0.2 mg/mL) were incubated with 0.9 mL of 0.2X JC-1 staining working solution. A time scan was performed directly using a fluorescence microplate reader (Gemini EM Microplate Reader, Molecular Devices, Sunnyvale, CA, USA) with an excitation/emission wave length of 485/590 nm, and observed under an Olympus BX5 fluorescence microscope imaging system (Olympus America, Melville, NY, USA).

The terminal transferase biotinylated-dUTP nick end labeling (TUNEL) method was performed to label cells undergoing apoptosis following the manufacturer’s instructions (Roche Applied Science, Penzberg, Germany). Briefly, the brain sections were incubated in a permeabilization solution and then incubated with a TUNEL reaction mixture. Finally, the sections were incubated with 10 μg/mL Hoechst 33342 in a humidified dark chamber. After the TUNEL method, the sections were stained with DAPI (4,6-diamidino-2-phenylindole) and mounted with Fluoromount (SouthernBiotech, Birmingham, AL, USA), and FITC-labeled apoptotic cells were then imaged on an Olympus BX5 fluorescence microscope imaging system (Olympus America, Melville, NY, USA). The number of apoptotic neural cells per view was counted using microscopy at × 400 magnification.