Culturing of HEK293 and COS-7 cells and immunolabeling were essentially as described [42 (link)]. HEK293 and COS-7 cells were transfected using TurboFect (Thermo Scientific). Mitochondria of COS-7 cells were stained with 0.2 μM MitoTracker Deep Red FM (Molecular Probes) in medium at 37°C for 1 h and cells were subsequently fixed with 4% PFA for 7 min.
Primary rat hippocampal neuron cultures for immunofluorescence analyses were prepared, maintained, and transfected (at DIV4) as described previously [16 (link),43 (link),44 (link)]. Fixation was done at DIV6 in 4% (w/v) PFA in PBS pH 7.4 at RT for 4–6 min. Permeabilization and blocking were done with 10% (v/v) horse serum, 5% (w/v) BSA in PBS with 0.2% (v/v) Triton X-100. Phalloidin stainings and antibody incubations were done in the same buffer without Triton X-100 according to [42 (link),44 (link)].
Primary rat hippocampal neuron cultures for immunofluorescence analyses were prepared, maintained, and transfected (at DIV4) as described previously [16 (link),43 (link),44 (link)]. Fixation was done at DIV6 in 4% (w/v) PFA in PBS pH 7.4 at RT for 4–6 min. Permeabilization and blocking were done with 10% (v/v) horse serum, 5% (w/v) BSA in PBS with 0.2% (v/v) Triton X-100. Phalloidin stainings and antibody incubations were done in the same buffer without Triton X-100 according to [42 (link),44 (link)].
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