Bx50 fla

Four weeks after neonatal hypoxia, the ultrastructural changes in synapses in the hippocampus and cerebral cortex were evaluated by TEM as described previously [34 (link)]. The cerebral tissues were dissected into 1 mm³ tissue blocks, and fixed in 2% glutaraldehyde at 4°C for 2 h. The tissue was rinsed in cacodylate buffer, postfixed with 1% osmium tetroxide for 2 h, and then dehydrated in a graded ethanol series. Subsequently, the tissue was infiltrated with a mixture of one-half propylene oxide overnight and embedded in resin. After that, 70 nm sections were stained with 3% uranyl acetate for 20 min and 0.5% lead citrate for 5 min. Five pictures of each subregion per ultrathin section were taken at 9700x or 37000x magnification. The number of synapses, thickness of the postsynaptic density (PSD), and width of the synaptic cleft were measured using ImageJ software (BX50-FLA, Olympus, Japan).

Chromosomal condensation and morphological changes in the nucleus of PC12 cells were observed using the chromatin dye Hoechst 33258. The PC12 cells were fixed with 4% paraformaldehyde in 0.1 M phosphate buffered saline (PBS) for 10 min. After three rinses with PBS, the cells were stained with 5 mg/L Hoechst 33258 for 10 min. Slides were rinsed briefly with PBS, air dried, and then mounted in an anti-fluorescein fading medium (Perma Fluor, Immunon, PA, USA). Slides were visualized under a fluorescent microscope (BX50-FLA, Olympus, Tokyo, Japan). Viable cells displayed normal nuclear size and uniform fluorescence, whereas apoptotic cells showed condensed nuclei or nuclear condensations.

To examine mPTP opening, an mPTP fluorescence assay (Genmed Scientifics Inc., MA, USA) was performed. This method has been previously described [29 (link)]. In brief, H9c2 cells were loaded with 8 mM cobalt chloride and 0.25 mM calcein-acetoxymethylester (calcein-AM) at 37°C for 20 min. The fluorescence signal was observed by using a fluorescence microscope (Olympus, Bx 50-FLA) at 488 nm excitation and 525 nm emission. The results are presented as relative fluorescence intensity. The average fluorescence intensity was analyzed by Image-Pro advanced software.

H9C2 cells were fixed with ice-cold 4% paraformaldehyde for 20 min after washed with 0.1 mol/L sodium cacodylate buffer. Next, the cells were washed three times with 0.1 mol/L sodium cacodylate buffer before stained with Hoechst 33258 (Beijing Solarbio Science & Technology Co., Ltd., China) for 15 min in the dark. Finally, an inverted fluorescence microscope (Olympus; BX50-FLA; Japan) was applied to visualize the apoptotic cells.

When HUVECs were cultured to 80%–90% confluence in a six-well plate, a line was gently drawn on the bottom of each well with a pipette and washed three times with PBS. The width of the scratch was measured after different treatments. The degree of the scratch healing was observed under an inverted microscope (BX50-FLA, Olympus, Tokyo, Japan).