Fluoview 3000rs confocal microscope

Cells were grown on coverslips 24 h before transfection and treatment with CCCP. Immunostaining were performed as previously described^{67 (link)}. Cells were washed with PBS, fixed with 3.5% paraformaldehyde in PBS for 10 min, permeabilized with 0.15% Triton X-100 in PBS for 15 min, and blocked in 2% BSA in PBS for 30 min. Primary antibodies were used at 1:200 for 1 h at room temperature, followed by secondary antibodies at 1:1,000 for 1 h at room temperature. Final washing included incubation with 500 nM Hoechst (Sigma-Aldrich) for 10 min. Cells were mounted with Slow Fade (Molecular Probes). Images were acquired by Olympus FluoView 3000RS confocal microscope with 20x/0.75 objective in the UNC Neuroscience Microscopy Core facility using Olympus FluoView (FV31S-SW) software. For each individual experiment, quantification of Parkin translocation was estimated by counting a minimum of 3 × 100 cells in 3 fields of view for each treatment.

Except for ChAT microscopy, all other imaging was done by using the oil immersion 63 X objective on the Leica SP8 confocal microscope and by using the oil immersion 40 X objective on the Olympus Fluoview 3000rs confocal microscope with resonance scanning mode. For thick tissues, such as human tissues, the Galvano mode of the Olympus Fluoview 3000rs microscope that enabled higher resolution imaging and averaging was used. Images obtained were then analyzed using Fiji (https://fiji.sc/).
Live tissue imaging of the mouse gut tissue was performed by harvesting small intestinal tissue from an adult Wnt1-Cre:Rosa26^lsl-tdTomato mouse and immediately putting it in OptiMEM solution. The tissue was then immediately put on a chamber slide containing OptiMEM and imaged in live tissue culture conditions of the EVOS M7000 microscope under the 20 X objective.

Glass slides were coated with Poly‐ornithine for 20 min at 37°C. They were then washed with H20 Milli‐Q and PBS. CLL primary cells were seeded with a concentration of 1 × 10⁶ cells/slide and let adhere for 2 h. PFA4% was used to fix cells for 15 min and PBS washes were then made. Cells were permeabilized and blocked with a solution of 10% FBS, 0,3% Triton x‐100 (Sigma‐Aldrich) and 1 mg/mL of BSA. Fluorescence staining were done by leaving primary antibodies o/n at 4°C, secondary antibodies 2 h at room temperature and DAPI (1:2000) for 5 min. Then slides were mounted with ProLong Gold Antifade Reagent (Invitrogen) and let dry for 24 h. Images were acquired on Olympus FluoVIEW 3000 RS Confocal microscope (Olympus) with a 30× (NA 1.05) silicone objective. Primary antibodies used: PYK2 (Cell Signalling, 1:200), pPYK2 (Y402) (Cell Signalling, 1:200), FAK (Cell Signalling, 1:200) and pFAK (Y397) (Thermo Fisher, 1:200). Secondary antibodies: Alexa 488 (1:500). Ten images for each condition and patients were acquired and analysed with Fiji ImageJ. A macro was created to measure the mean grey value of the fluorescence signal of the different markers in order to obtain a quantification of the levels of expression of the protein of interest. Graphs and statistical analysis were performed using GraphPad Prism (https://www.graphpad.com/scientific‐software/prism/).