Goat anti rabbit antibodies

Human U2OS cells were cultured in DMEM containing high-glucose and GlutaMAX (Thermo Fisher Scientific) supplemented with 100 U/mL penicillin and 100 $\mu$ g/mL streptomycin (Merck Millipore), 1 mM sodium pyruvate (Sigma-Aldrich), and 10% (vol/vol) FBS (Merck Millipore) at 37 °C and 5% ${\text{CO}}_2$ . Cells were seeded on sapphire crystal discs (Engineering Office M. Wohlwend GmbH) and grown overnight. Cells were fixed with prewarmed 4% formaldehyde in PBS (137 mM NaCl, 2.68 mM KCl, and 10 mM ${\text{Na}}_2$ ${\text{HPO}}_4$ , pH 7.4) for 10 min at 37 °C, extracted with 0.5% (vol/vol) Triton X-100 in PBS, blocked with 5% (wt/vol) BSA in PBS, and incubated with polyclonal antibodies against the mitochondrial protein Tom20 (Santa Cruz Biotechnology) and vimentin (Sigma-Aldrich) for 1 h. After five washing steps with PBS and blocking with 10% (wt/vol) BSA in PBS, primary antibodies were detected with secondary sheep anti-mouse Alexa Fluor 488 antibodies (Invitrogen) or goat anti-rabbit antibodies (Jackson ImmunoResearch Laboratories) custom-labeled with Alexa Fluor 594 (Life Technologies) for 1 h. Samples were washed five times with PBS and covered with PBS containing 2.5 $\mu$ g/mL DAPI (Sigma-Aldrich). The cells were plunge-frozen in liquid nitrogen-cooled propane. Samples were stored submerged in liquid nitrogen for later imaging.