Anti cytochrome c a 8

Western blot was performed as described previously [87 (link)]. Lysates of HMDMs or their EVs were prepared in RIPA buffer supplemented with cOmplete Protease Inhibitor Cocktail (Roche, Basel, Switzerland). Anti-Alix (1A12, Santa Cruz Biotechnology, Dallas, Texas, USA), anti-CD63 (Santa Cruz Biotechnology), anti-Calnexin (AF18, Santa Cruz Biotechnology), anti-cytochrome c (A-8, Santa Cruz Biotechnology), and anti-TLR7 (4F4, sc-57463, Santa Cruz Biotechnology) were used as primary antibodies.

Hippocampi were lysed in extraction buffer (50 mM sodium acetate, 1M NaCl, 0,1% Triton X100, pH 4,0, protease inhibitor tablets), and by means of a Sonifier (UP50H Hielscher) with a MS1 sonotrode. Cell lysates were cleared at 20.000× g for 10 min at 4°C. Proteins were blotted on Immun-Blot PVDF membranes (Biorad). Blocking and antibody incubation were performed in 10% goat serum, 5% milk powder (Biorad) for 3–4 h. For BDNF detection, rabbit anti-BDNF 17H (1/6000, provided by Michael Sendtner, Institute for Clinical Neurobiology, Würzburg, Germany) was incubated over night at 4°C. Anti-Cytochrome C A-8 (Santa Cruz, 0.2 μg/ml) was used as loading control. For detection, the ECL Plus kit (GE Healthcare) was used in combination with horseradish peroxidase-coupled secondary antibodies (Jackson Laboratories).