Zen 2012 sp2

The scaffold-CAM complex was fixed in 4% paraformaldehyde (PFA) at 4°C overnight. The cell-seeded constructs were excised from the CAM, embedded in paraffin (Haslab GmbH, Ostermundigen BE, Switzerland) and sectioned vertically in 5 μm thick sections. For histological evaluation, sections from the center of the scaffolds were stained with Hematoxylin and Eosin (H&E) (Mayer's hemalum solution, Merck KGaA, Darmstadt, Germany; Eosin Y, Sigma-Aldrich Chemie GmbH, Buchs SG, Switzerland). Pictures were taken using the Axio Scan.Z1 slidescanner (Carl Zeiss AG, Oberkochen, Germany), a Hitachi HV-F202FCL camera (Hitachi, Ltd., Tokyo, Japan), and the ZEN 2012 SP2 software (Carl Zeiss AG, Oberkochen, Deutschland) provided by the Center for Microscopy and Image analysis, University of Zurich.

Testes or antennae were squashed on 12 diameter round coverslip with a 44 × 60 mm overlaying coverslip. Immunofluorescence protocols were the same as above using PFA. Images were acquired using the Elyra PS.1 system from Zeiss (Carl Zeiss, AG, Jena) equipped with a PCO edge 5.5 camera and the ZEN 2012 SP2 software (black edition). The objective used for all acquisitions is a Plan-apochromat 63 × 1.4 NA.

FSK cells, 5 × 10⁵, were cultured on 35 mm glass bottom dishes (MatTek Corporation, MA, USA) for 24 h in the presence and in the absence of 250 μM FA. The cells were then washed, fixed with 4% paraformaldehyde (PFA), blocked with 3% BSA (Sigma-Aldrich) and permeabilized with 0.25% Triton X-100 (Sigma-Aldrich). Immunolabeling was carried out using anti-TOMM20 Alexa Fluor® 647-conjugated (1:500), anti-53BP1 Alexa Fluor® 488-conjugated, anti-PINK1 FITC-conjugated (Abcam). CCCP at 1 µM final concentration was used as positive control for the recruitment of PINK1 to the mitochondria. DNA was visualized using an anti-DNA primary antibody (Abcam) (1:200) with IgM-PE as secondary antibody (1:1000). DNA DBS breaks were also monitored using a phospho-H2AX (S139) FITC-conjugated antibody and etoposide (Sigma) at 50 nM final concentration was used as positive control for inducing DNA DBSs in the mitochondrial DNA. Nuclei were counterstained with NucBlue® Fixed Cell Stain Ready Probes™ (Life Technologies) and all IF images were acquired with 60× oil immersion objective on the Zeiss LSM780 controlled by Zen 2012 SP2 software (Carl Zeiss MicroImaging). Each staining was performed in duplicate and repeated two times using different cell batches.

Group data were analysed in SigmaPlot 13.0 using one-way ANOVAs, where p < 0.05 was taken as the significance threshold and expressed as mean ± SEM. Tests for normally distributed data are built into the SigmaPlot software. Confocal images were captured and analysed with Zen 2012 SP2 (Zeiss) software. Oocyte concentration-response data were fit to a Hill equation to obtain an EC₅₀ and Hill co-efficient for each oocyte recording. These parameters were then averaged across multiple oocyte experiments of the same type. Single channel recordings were analysed in QuB software. Single channel currents were idealised and separated into discrete activations by applying critical shut times of 50 ms in glutamate alone or 50–100 ms in glutamate plus IVM to separate and define single receptor active periods. Patches yielded between 30–250 individual active periods. Critical shut times were determined by generating an initial shut duration histogram to continuous data that included inactive periods corresponding to receptor desensitisation. The selected critical times eliminated periods of receptor desensitisation while retaining singe receptor active periods. Mean active period duration and intra-activation open probability was estimated from each patch. Group means were obtained by averaging across multiple patches for each recording condition.