Anti cytochrome c antibody

Following treatment, growth medium containing 100 nM MitoTracker probe® (Molecular Probes, Eugene, OR, USA) was added to the culture, which was incubated for 30 min at 37°C. The cells were washed with PBS and then fixed in 4% formaldehyde in PBS for 15 min at 37°C. After washing the cells, they were permeabilized with 0.1% Triton X-100 in PBS for 15 min at 4°C. To prevent nonspecific binding, the cells were incubated in blocking buffer (1% BSA in PBST, pH 7.4) for 30 min at room temperature. Anti-cytochrome c antibody (0.5 μg/mL; 2 mL) (Abcam, Cambridge, MA, USA) was added and incubated for 1 h at room temperature. The coverslip was washed and then incubated in 2 mL Hoechst 33258 stain (1 μg/mL in PBS) (Sigma-Aldrich, St. Louis, MO, USA) for 20 minutes. The coverslip was washed again before being mounted with a drop of mounting medium (90% glycerol in PBS) and sealed with nail polish and stored in the dark at 4°C. The slide was examined on the Axioplan 2 fluorescence microscope (Zeiss, Jena, Germany) at 630x magnification and micrographs of the cells were taken using AxioVision 4.8 software. The excitation and emission wavelengths (nm) of the fluorescent dyes were as follows: Alexa-488, excitation: 499 and emission: 519; MitoTracker orange probe, excitation: 554 and emission: 576; and Hoechst 33258, excitation: 343 and emission: 483.

The serum biochemical reagents included ALT (LOT: 140121005), AST (LOT: 140221004), ALP (LOT: 140321002), TC (LOT: 141621013), TG (LOT: 141721003), LDL (142021004), and HDL (LOT. 142121006). TNF-a (E-EL-R2856-96T), IL-6 (E-EL-R0015-96T), and IL-1β (E-EL-R0012-96T) ELISA kits were purchased from Elabscience Biotechnology Co., Ltd. Anti-Caspase-8 antibody (Abcam, ab108333), anti-Caspase-3 antibody (Abcam, ab184787), anti-Cytochrome C antibody (Abcam, ab133504), and anti-IKK-β antibody (Abcam, ab124957) were purchased from Abcam. BCL2 monoclonal antibody (60178-1-1 g) and GAPDH (60004-1-Ig) antibody were obtained from Proteintech Group, Inc. NF-κB p65 antibody, phospho-NF-κB p65 antibody (LOT:17), a-SMA (CST, #19245), IκB-α antibody (CST, #4812), and phospho-IκB-α antibody (CST, #2859) were provided by Cell Signaling Technology, Inc. Chemiluminescence ECL Detection Kit (P90719) was obtained from Bobst Biotechnology.

Blastocysts were stained for mitochondria according to the method descried in the previous study [32 (link)]. Briefly, blastocysts were incubated in IVC medium supplemented with 0.5 μmol/L MitoTracker Red CMXRos (Molecular Probes, Eugene, OR) for 30 min in incubator with 5% CO₂ for 30 minutes at 38.5°C, followed by three washes with dPBS/PVA for 20 minutes. Then the blastocysts were fixed in 3.7% (w/v) paraformaldehyde in dPBS/PVA at room temperature. After 1h of permeabilization in PBS containing 0.1% Triton X-100 at 38.5°C, blastocysts were then blocked with 3% BSA in dPBS for 1h. They were then labeled with 100 μg/ml anti-cytochrome c antibody (Abcam) in blocking solution overnight in 4°C then labeled by rhodamine labeled second antibody. Nuclear was staining with 10 μg/ml Hoechst 33342 in PBS for 10 minutes, followed by washing three times in PBS, and then mounted on glass slides. Blastocyst were observed with a laser-scanning confocal microscope (Zeiss LSM 710 META, Germany). Co-localization of mitochondrial and cytochrome c were evaluated by Person’s Correlation coefficient.

The S. aspratus fruiting bodies were obtained from Yimeng Yisheng edible fungus cooperative (Yunnan, China). Human uterine cervix carcinoma cell line (Hela), human hepatoma cell line (HepG-2), human stomach cancer cell line (HGC-27), and human normal liver cell line (MRC-5) were purchased from the cell bank of Shanghai Institute of Cell Biology (Shanghai, China). Sepharose CL-4B gel was obtained from Amersham (Uppsala, Sweden). Dextran standards, standard monosaccharides, 5-fluorouracil (5-Fu), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide (MTT), 5,5′,6,6′-tetraethylimida-carbocyanine iodide (JC-1), and poly(2-hydroxyethylmethacrylate) (poly-HEMA) were obtained from Sigma (St. Louis, MO, USA). Fetal bovine serum (FBS) and RPMI-1640 medium were obtained from Wisten Biotechnology (WISTEN Co. Ltd., Nanjing); 24-well transwell plates were acquired from Corning (NY, USA). Anti-β-actin antibody, anti-caspase-9 antibody, anti-caspase-3 antibody, anti-Bcl-2 antibody, anti-Bax antibody, and anti-cytochrome c antibody were purchased from Abcam (Cambridge, UK). All the kits used in assays were supplied by Shanghai Beyotime Bioengineering Institute (Shanghai, China). All other chemical reagents used were of analytical grade.

Before immunoflorescent staining for cytochrome c, MitoTracker® Red CMXRos (Invitrogen, M7512) was chosen to staining mitochondria membrane firstly. In details, RPMI-8226 cells were infected with different viruses at an MOI of 4. After 48 hours, collecting cells and stained with prewarmed (37 °C) MitoTracker® probe (100–500 nM) for 15–45 minutes. Immunoflorescent staining for cytochrome c was performed as described previously46 (link). In details, cells were washed with PBS, and then fixed with 4% paraformaldehyde. After permeablized by 0.3% Triton X-100 and incubated with goat serum, cells were stained with anti-cytochrome c antibody (Abcam, 1:200 dilution) overnight at 4 °C. Then, cells were incubated with a goat anti-rabbit antibody as secondary antibody (1:500) at 37 °C for 1 h and 1 μM DAPI (SouthernBiotech, USA) for 10 min. Finally, samples were examined with a Nikon confocal microscope (Nikon C1-Si, Japan), and images were processed using NIS-Elements software package.

3-(4,5-Dimethylthizol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and MG (40%, w/v), Propidium Iodide (PI) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Luteolin (purity > 98%) was purchased from Shanxi Sciphar Biotechnology Co., Ltd (Shanxi, China). Fetal bovine serum (FBS) and Dulbecco’s modified Eagle medium (DMEM) were obtained from Gibco (Grand Island, NY, USA). mTOR, phospho-mTOR (Ser2448), phospho-4E-BP1 (Thr37/46), AKT, phospho-AKT (Ser473), phospho-p70S6K (Thr389) were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-Cytochrome C antibody was purchased from Abcam (Cambridge, UK). Antibodies against Bax and Bcl-2 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse anti-β-actin monoclonal antibody was purchased from Abmart (Shanghai, China). All secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).