Tom20 antibody

For H&E staining, tissues were fixed with 10% formalin and paraffin-embedded, and then standard procedures were performed. To label blood vessels in vivo, Fluorescein labeled Griffonia Simplicifolia Lectin I (4 µg per mouse, Vector Laboratories, Cat# FL-1101) was gently injected into 2-month-old male mice via tail vein. After 20 min, the mice were sacrificed and iWAT tissues were isolated and carefully minced with scissors. The iWAT tissues were washed twice with pre-warmed PBS and cover-slipped by Fluoromount-G (SouthernBiotech, Cat#0100-01). For immunofluorescence staining, formalin-fixed and paraffin-embedded tissue sections were deparaffinized and rehydrated through graded ethanol solutions. After pre-incubation with a blocking buffer (PBS containing 5% normal goat serum and 0.3% Triton X-100) for 60 min, slides were incubated with Ucp1 antibody (Sigma, Cat#U6382) (1:500 dilution), Tom20 antibody (Santa Cruz, Cat#sc-17764) (1:100 dilution), or CD31 antibody (1:250) (Millipore Cat#MAB2148-C) in blocking buffer at 4 °C overnight. Subsequently, the slides were washed, and incubated with Alexa Flour 488-conjugated or 594-conjugated secondary antibody and DAPI for 60 min. Images were acquired and processed with the same setting for transgenic mice or knockout mice and control littermates.

All chemicals including cell culture medium (Dulbecco´s modified Eagle´s medium (DMEM)) as well as the catalase-polyethylene glycol (PEG-catalase) were obtained from Sigma or Merck (Darmstadt, Germany) unless otherwise stated. The fetal bovine serum was from Pan Biotech (Aidenbach, Germany). The Protein Assay Kit (Bio-Rad DC, detergent compatible) was from Bio-Rad Laboratories (Feldkirchen, Germany). The enhanced chemiluminescence system (SuperSignal West Pico/Femto Maximum Sensitivity Substrate) was supplied by Pierce (Fisher Scientific, Schwerte, Germany). Penicilin/Streptomycin was obtained from Biochrom (Berlin, Germany) and Glutamax from Gibco (Darmstadt, Germany). MitoTEMPO was purchased from Enzo Life Sciences (Lausen, Schweiz). MitoSOX Red and MitoTRACKER Green FM were obtained from Fisher Scientific (Schwerte, Germany). Beta-actin antibody was purchased from Cell Signaling (Massachusetts, USA). The polyclonal rabbit α-hapten antibody directed against dimedone tagged sulfenic acids was kindly provided from K.S. Carroll [35 (link)]. TOM-20 antibody was purchased from Santa Cruz Biotechnology (Dallas, USA). DMSO was obtained from Roth (Karlsruhe, Germany). Horseradish peroxidase (HRP) conjugated goat anti-rabbit IgG from Dianova (Hamburg, Germany) and HRP conjugated rabbit anti-mouse IgG from Dako (Glostrup, Denmark) were used as a secondary antibody.

The samples obtained were separated under denaturing conditions in 12.5% SDS-PAGE and transferred to a nitrocellulose membrane (0.2 μm pore). Precision Plus Pre-stained Standards from Bio-Rad Laboratories (Hercules, CA, USA) were used as markers. After overnight blocking, each membrane was incubated with the appropriate primary antibody. The polyclonal TSPO antibody (1:1000), monoclonal VDAC 1, and polyclonal VDAC 3 antibody (1:1000) were obtained from Abcam (Cambridge, UK); the monoclonal VDAC antibody (1:1000) was purchased from Calbiochem (San Diego, CA, USA); the polyclonal rabbit phospho-Akt (Ser473), phospho-GSK-3β (Ser9), and Akt antibodies (dilution 1:250) were obtained from Cell Signaling (Leiden, Netherlands). The monoclonal anti-CNP antibody (anti-CNP Ab) was obtained as described [43 (link)] and used at a 1:10,000 dilution, the monoclonal GSK3β antibody was used at a 1:2000 dilution, and the Tom 20 antibody (Santa-Cruz, Dallas, TX, USA) served as a loading control and was used at a 1:2000 dilution. Immunoreactivity was detected using the appropriate secondary antibody conjugated with horseradish peroxidase (Jackson Immuno Research, West Grove, PA). Peroxidase activity was detected using Enhanced chemiluminescence (ECL) reagents (Bio-Rad, Hercules, CA, USA).

Immunoblot analysis was performed as we described previously30 (link). Rabbit polyclonal anti-ANT-1 antibody and rabbit monoclonal anti-β-actin antibody were from Sigma-Aldrich (St Louis, MO, USA, SAB2105530, 1:1,000) and Cell Signaling (Danvers, MA, USA, 4970, 1:5,000), respectively. Rabbit polyclonal anti-ANT2 antibody was generated by immunization with KLH-conjugated N-terminus ANT2 peptide (NH-TDAAVSFAKDFLAG-COOH) at Lampire Biological Laboratories (Ottsville, PA, USA) and purified with protein G (GE Healthcare, Buckinghamshire, UK) and antigen-conjugated columns. The ANT2 antibody was diluted to 1:200 as being used. COX IV (4850, 1:1,000) and VDAC (4866, 1:1,000) antibodies were from Cell Signaling (Danvers, MA, USA). Tom20 antibody was from Santa Cruz Biotechnology (sc-11415, 1:500), Catalase and SOD2 antibodies were from Sigma (St. Louis, MO, USA, C0979, 1:1,000) and Abcam (Cambridge, UK, ab16956, 1:1,000), respectively. Uncropped scans of the critical immunoblots are shown in Supplementary Fig. 6.

SKOv3-ARHI cells were seeded into a 75 cm² flask and allowed to reach confluence. Cells were treated with 1 μg/mL of Doxycycline for different time points as previously discussed. After treatment, cells were trypsinized and harvested at 500xg for 10 min. Cells were then fixed in 3 % formaldehyde and permeabilized with 0.1 % Triton X-100. Cells were then counted, aliquoted and incubated in 2 μg of TOM20 antibody (Santa Cruz Biotechnology) for 40 min. Cells were then washed and incubated in F(ab’) 2-Goat anti Rabbit IgH (H + L) Secondary Antibody, Alexa Fluor® 488 conjugate (Thermo Scientific).