A2153

Formalin-fixed paraffin-embedded sections were deparaffinized with Histoclear (National Diagnostics) and rehydrated in a series of EtOH solutions. Antigen retrieval was performed in a microwave for 20 min in a 10 mM Citrate, 0.05% Tween-20, pH 6.0 solution. Sections were blocked in 3% bovine serum albumin (BSA) (Sigma) for 1 h, followed by overnight incubation in the primary antibody diluted in 3% BSA (Sigma, A2153). For immunofluorescence, detection and labeling were performed with secondary antibodies conjugated to Alexa-Fluor-488 or Alexa-Fluor-594 fluorophores (Invitrogen, A21203, A21206, or A21209) at a dilution of 1:250 in 3% BSA. For peroxidase staining, sections were washed and incubated in Biotinylated secondary antibodies (Goat Anti-Rabbit IgG Biotinylated, Vector Biolabs, BA-1000 at 1:200 dilution in 3% BSA; Goat Anti-Rat IgG Biotinylated, Vector Biolabs, BA-9400, 1:200 dilution in 3% BSA; Vector Mouse on Mouse Immunodetection Kit, Vector Biolabs, FMK-220 following manufacturer’s conditions), followed by avidin/biotin complex formation (Vectastain ABC, PK-6100, Vector Biolabs). Sections were then incubated with DAB peroxidase (horseradish peroxidase) substrate (Vector labs, SK-4100) and counterstained with hematoxylin. Immunofluorescence images were obtained using a Zeiss LSM 880 confocal microscope. All antibodies and dilutions are listed in Supplementary Table 3.

Immunocytochemistry was carried out as described previously^{56 (link)}. Cells were fixed with 4% paraformaldehyde/PBS for 15 min, permeabilized, and blocked with PBS containing 3% bovine serum albumin (A2153, Sigma-Aldrich) with 0.1% Triton X-100 (T9284, Sigma-Aldrich). Subsequently, cells were labeled with the primary ab, anti-MAP1LC3B (1/500) or anti-pH2AX (1/250). For negative controls, the primary ab was replaced with normal rabbit IgG or normal mouse IgG. After washing out the primary ab, cells were secondarily stained with Alexa Fluor 488 donkey anti-rabbit IgG (1/1000) or Alexa Fluor 488 donkey anti-mouse IgG (1/1000), followed with Hoechst33342 nuclear staining for 10 min. Cells were observed using a confocal microscope (LSM700, Carl Zeiss, Oberkochen, Germany).

HeLa cells, grown on borosilicate cover glasses (Thermo Fisher Scientific), were co‐transfected with mCherry‐Parkin (Addgene, 23956) and either mitochondria‐targeted GFP or one of the GFP‐tagged IF₁ clones (wild type, dominant negative E30A, or constitutively active H49P) as previously demonstrated (Faccenda et al., 2017 (link)). After 48 h from transfection, cells were fixed with 4% paraformaldehyde in PBS (10 min) and prepared for immunostaining. Briefly, cells were permeabilized in 0.1% Triton X‐100 in PBS for 15 min and incubated in blocking solution (10% normal goat serum, Thermo Fisher Scientific, PCN5000; 3% BSA, Sigma‐Aldrich, A2153; 0.01% Triton X‐100 in PBS) for 1 h. Cells were then incubated overnight with an anti‐ATPβ (Abcam, ab14730) 1:500 in blocking solution, washed 5 times in PBS, and incubated for 2 h with an Alexa Fluor 647‐conjugated anti‐Mouse IgG1 (Thermo Fisher Scientific, A21240) 1:500 in blocking solution. After a further washing (5 times in PBS), cells were mounted on slides using mounting medium with DAPI (Abcam, ab104139).

Cells were plated at 20,000 cells/well on coverslips in 24 well plates and allowed to attach overnight. Wells were fixed, permeabilized, and blocked as described previously [54 ]. Cells stained on cover slips were mounted on slides using Fluorescence Mounting Medium (S3023, DAKO). The coverslips were then incubated for 16 hours at 4°C with polyclonal rabbit anti-CK19 (10 μg/ml, ab15463, Abcam) or polyclonal rabbit anti-N-cadherin (1:100, ab76057, Abcam). Alexa Fluor 594 polyclonal goat anti-rabbit (1:700, A-11012, Life Technologies) was used as a secondary antibody in 1% BSA (A2153, Sigma-Aldrich) in PBS for 1 hour at room temperature. For L1CAM, monoclonal anti-L1CAM-PE (1:50, ab95694, Abcam) was used. Nuclei were counterstained with DAPI (1 μg/ml, 10236276001, Roche,), in PBS for 5 minutes at room temperature. Images were obtained using an Axiovert 200M fluorescence microscope (Zeiss Axiovert 200 M, Carl Zeiss MicroImaging GmbH, Jena, Germany) with CCD camera (Zeiss Axiocam HR), using Axiovision software. All images are taken with 40x magnification.

Cells were plated and cultured on 0.1% gelatin (CA087-100, GenDepot, TX, USA)-coated coverslips. Paraformaldehyde (4%) was used to fix the cells for 15 min at room temperature. After thorough washing with phosphate-buffered saline (#10010-023, Thermo Fisher Scientific, MA, USA), cells were then permeabilized with ice-cold methanol for 10 min at − 20 °C. Blocking was performed with 3% bovine serum albumin (A2153, Sigma Aldrich, MO, USA) for 1 h. Primary antibodies used in this study were as follows: OCT3/4 (1:200; sc-5279, Santa Cruz Biotechnology, TX, USA), SIRT2 (1:100; S8447, Sigma-Aldrich, MO, USA), β-III-tubulin (1:1000; ab41489, Abcam, Cambridge, UK), α-smooth muscle actin (1:200; A5228, Sigma Aldrich), and SOX17 (1:50; ab191699,Abcam, Cambridge, UK). As secondary antibodies, anti-chicken Alexa Fluor 647 (ab150171, Abcam, Cambridge, UK), anti-mouse FITC (ab6785, Abcam, Cambridge, UK), and anti-rabbit TRITC (ab6718, Abcam, Cambridge, UK) were used at a dilution of 1:500. Nuclear counterstaining was performed with DAPI (D9542, Sigma Aldrich, MO, USA) for 10 min.