Lsm 710 meta

To immunostain oocytes for Plk1 or alpha-tubulin, the cells were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) and then transferred to a membrane permeabilization solution (0.5% Triton X-100) and kept there for 1 hr. After 1 hr in blocking buffer (PBS containing 1% bovine serum albumin), the oocytes were incubated overnight at 4 °C with an anti-human Plk1 antibody (Santa Cruz Biotech, Santa Cruz, CA, USA). After three washes with PBS containing 0.1% Tween 20 and 0.01% Triton X-100, the oocytes were incubated with an Alexa Fluor 488-conjugated goat anti-mouse IgG (1:100 dilution) for 1–2 h at room temperature. For staining of the cytoplasmic actin mesh or cortical actin, the oocytes were fixed and stained with phalloidin-tetramethylrhodamine (10 μg/mL), which labels F-actin. For staining with an anti-α-tubulin-fluorescein isothiocyanate antibody (1:200 dilution), the oocytes were incubated with this reagent for 1 h, washed three times in washing buffer for 2 min each time, incubated with Hoechst 33342 (10 μg/mL in PBS) for 15 min, and then washed three times in washing buffer. The samples were mounted onto glass slides and examined under a confocal laser scanning microscope (Zeiss LSM 710 META, Jena, Germany, with a 40× water immersion objective lens).

Mitochondrial fission was analysed by staining mitochondria as we and others have described earlier with some modification 29, 30. Briefly, cells were plated onto the coverslips coated with 0.01% poly‐l‐lysine. After treatment, they were stained for 20 min. with 0.02 μM MitoTracker Red CMXRos (Molecular Probes, Eugene, OR, USA). Mitochondria were imaged using a laser scanning confocal microscope (Zeiss LSM710 META, Dublin, CA, USA). To quantitatively analyse cells with mitochondria fission, those cells with disintegrated mitochondria were taken as mitochondrial fission. The percentage of cells with fragmented mitochondria relative to the total number of cells was presented as the mean ± S.E.M. of at least three independent experiments, counted by an observer blinded to the experimental conditions. A range of 100–150 cells in 20–30 random fields were counted.

After washing three times with PBS/PVA, embryos were fixed in 3.7% paraformaldehyde for 30 min at room temperature, permeabilized with PBS/PVA containing 0.5% Triton X-100 at room temperature for 30 min, and incubated in PBS/PVA containing 1.0% bovine serum albumin at room temperature for 1 h. These embryos were incubated overnight at 4 °C with anti-LC3 (1:100; Cat #ab58610, Abcam, Cambridge, UK), anti-cytochrome c (1:100; Cat #ab110325, Abcam, Cambridge, UK), and anti-γH2A.X (1:100; Cat #2577, Ser139, Cell Signaling Technology, Danvers, MA, USA) diluted 1:100 in blocking solution. After washing three times with PBS/PVA, the embryos were incubated at room temperature for 1 h with goat anti-rabbit IgG, rabbit anti-goat IgG, or anti-mouse IgG. The oocytes and embryos were stained with 10 µg/mL Hoechst 33342 for 5 min, washed three times with PBS/PVA, mounted onto slides, and examined using a confocal microscope (Zeiss LSM 710 META). Images were processed using Zen software (version 8.0, Zeiss).

Following plating on gelatin-coated Lab-Tek I chambers, cells were maintained in phenol red-deprived DMEM-F12 (supplemented with low serum growth supplement and 10% FBS) for epifluorescence video microscopy experiments. For bright field video microscopy, cells were observed over a 30 min period using a Zeiss Axiovert 200M equipped with a Coolsnap camera (Roper Scientific, Trenton, NJ, USA). Cells were maintained at 37°C in 5% CO₂ atmosphere using a temperature-controlled chamber (PECON, Erbach, Germany). Data were acquired using MetaMorph software (Roper Scientific). Timespan between image capture was 4 s. Confocal microscopy images of fixed cells were collected with a Zeiss (Oberkochen, Germany) LSM710 Meta confocal microscope using either a ×63 Plan Apochromat objective (oil immersion, 1.40 NA, DIC) at a 132 nm.pixel^-1 resolution, leading to a slight XY oversampling. Optical slices were spaced by 1 μm so as to limit Z oversampling. Fluorescence filters were chosen and tested for each combination of plasmid construction and fluorescent dye so as to abolish spectral overlap. Zen software program was used to acquire images.

C6 cells were treated with either LOB3 (0, 20, and 30 μM) or CytoB (5 μM) for the indicated time. For confocal microscopic analysis, the cells were fixed with 4% paraformaldehyde in PBS for 10 min and permeabilized with 0.5% Triton X-100 in PBS for 10 min. The cells were next blocked with 1% BSA in PBS for 1 h, followed by incubation with Rhodamine Phalloidin reagent or the antibodies specific for F-actin and cleaved caspase-3 at 4°C overnight. The cells were then incubated with Alexa Fluor 488-or 568-conjugated secondary antibodies for 1 h. The DNA of these cells was stained with Hoechst 33342 (10 μg/mL in PBS) for 30 min, followed by washing with PBS for 5 min three times. The cells were mounted on the glass slides and imaged using a laser-scanning confocal microscope (Zeiss LSM 710 META, Oberkochen, Germany) with a 63× oil-immersion objective lens.

Immunostaining was conducted as previously described^{41 (link)}. After washing thrice with PBS/PVA, blastocysts were fixed with 3.7% formaldehyde at RT for 30 min. Next, blastocysts were permeabilized in 0.5% Triton X-100 for 30 min at RT, and incubated in PBS/PVA containing 3.0% BSA at RT for 1 h. Subsequently, these blastocysts were incubated overnight at 4 °C with rabbit anti-GRP78 (1:100; ab21685, Abcam, Cambridge, United Kingdom), rabbit anti-HSF1 antibody (1:100; 12972S, Cell Signaling, Danvers, MA, United States), and rabbit anti-caspase3 antibodies (1:20; 9664S, Cell Signaling). After washing thrice with PBS/PVA, the blastocysts were incubated with Alexa Fluor 488™ goat anti-rabbit IgG (1:200; A32731, Invitrogen) or Alexa Flour 546™ donkey anti-rabbit IgG (1:200; A10040, Invitrogen, Carlsbad, CA, United States) at 37 °C for 1 h. After three washes, the blastocysts were incubated for 10 min with 5 μg/mL Hoechst 33342. Finally, the blastocysts were mounted on slides and examined under a confocal microscope (LSM 710 META; Zeiss, Oberkochen, Germany). Images were processed using Zen software (v.8.0; Zeiss, Jena, Germany) and then analyzed using ImageJ v.l.44 g software (National Institutes of Health, Bethesda, MD, USA).