Ipp version 6

Rat disc organ culture was carried out as we previously described (35 (link)). Ex vivo, discs were pretreated with 100 μM RV for 24 h or 2 mM NAC for 12 h or 100 μM PTIO for 12 h, then treated with or without 1 mM SNP for 18 h. Then the harvested discs were fixed in 4% paraformaldehyde, and then decalcified with EDTA for 2 weeks. After embedding with paraffin, 5-μm thick serial mid-sagittal sections of discs were made for slides. Mid-sagittal sections of discs were analyzed for apoptosis using in situ cell death detection kit according to manufacturer's instructions. DAPI staining was conducted for indication of total cells. TUNEL-positive apoptotic cells and DAPI-positive total cells of NP area on mid-sagittal sections of discs were identified by fluorescent microscope (IX71; Olmypus), and apoptosis rate was calculated as the percentage of numbers of TUNEL-positive cells to the numbers of total cells using IPP version 6.0 software (Media Cybernetics). The quantitative analysis was performed on three ×200 fields/section (three sections/disc and three discs for each kinds of treatment).

Mitochondrial membrane potential was measured with TMRM staining. NP cells were incubated with 100 nM TMRM at 37°C in dark for 30 min, washed with PBS 3 times and covered with fresh medium. NP cells were subsequently imaged using microscope (IX71, Olmypus). The excitation wavelength for TMRM was 549 nm. Three images (×200) of each kind of treatment were obtained for quantitative analysis of fluorescence intensity of the TMRM using IPP version 6.0 software (Media Cybernetics, Bethesda, MD, USA). The fluorescence intensity of the TMRM was expressed as mean density in IPP version 6.0 software.