710 confocal microscope

BY4742 yeast clones with human CI subunits that were cloned into the p340 construct were grown over night in 30 °C SD-His medium in 15 ml falcons with shaking, back diluted (OD₆₀₀ = 0.1) into 96 well plate and grown for 4 hours in 30 °C SD-His medium with shaking. Notably, the yeast were grown in 30 °C rather than in 37 °C to ovoid increased membrane propensity to be leaky, which likely influence growth and mask the influence of protein complementation on such growth. Then, yeasts were transferred into a 384 well plate for imaging in a Zeiss 710 Confocal Microscope. YFP expression was validated and the pattern of YFP distribution within the yeast cells were compared with the distribution patterns observed at Breker et al.^{41 (link)} to determine protein localization within the yeast.

Immunofluorescence microscopy was performed as described earlier. In brief, cultured NRVCs or cell lines were washed two times with 1× PBS and cells were fixed with 4% paraformaldehyde (PFA) in PBS and incubated at room temperature for 10 min. Fixed cells were washed two times with 1× PBS and permeabilized with 0.5% Triton X-100 at room temperature for 10 min. After washing two times with 1× PBS, cells were incubated with 0.1 mol/l glycine buffer (pH 3.5) at room temperature for 30 min. Cells were washed two times with 1× PBS and blocked with blocking buffer (1% bovine serum albumin, 0.1% Tween-20 in 1× PBS) for 1 h at room temperature. Cells were incubated with primary antibodies in blocking buffer at 4 °C overnight. Cells were washed three times with 1× PBS and incubated with Alexa-Fluor-conjugated secondary antibody (Life Technologies) for 1 h at room temperature. Cells were washed three times with 1× PBS and slides were mounted with Vectashield Hard Set mounting medium (Vector Labs), and allowed to dry at room temperature for 30 min. In some experiments, cells were stained with multiple primary antibodies. In those case, cells were re-blocked after the first secondary antibody treatment with blocking buffer for 30 min at room temperature. Stained cells were analyzed under 710 confocal microscope (ZEISS). Images were analyzed with the ImageJ software (NIH).

Oxygen Species Assay Kit (Beyotime, Shanghai, China) and MitoSOX Red (Thermo Fisher Scientific) were used to assess ROS levels in oocytes. Dichlorofluorescein (DCFH) is an oxidation-sensitive fluorescent probe. Oocytes were treated with or without cisplatin (1 or 20 μM) for 4 h and then incubated in M2 medium with 10 μM DCFH diacetate (DCFHDA) for 30 min at 37 °C. All three groups of oocytes were washed separately three times in fresh medium and images were captured on a scanning confocal microscope. For MitoSOX staining, cisplatin treated and non-treated oocytes were incubated in M2 medium containing 5 μM MitoSOX Red for 10 min at 37 °C in the dark. After washing three times in fresh medium under low light, oocytes were imaged under a Carl Zeiss 710 confocal microscope. In each experiment, fluorescence signals were acquired by confocal microscopy with the same scanning settings. ImageJ software (NIH Image, Bethesda, MD) was used to quantify fluorescence intensity as previously reported [21 (link)]. Briefly, fluorescence channels of the confocal images were separated and converted to 8-bit images, and then a threshold was set for each channel of fluorescence. The mean fluorescence intensity of thresholded fluorescence images of different groups was calculated using the measurement function in ImageJ software.