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, After permeabilization with 0.1% of saponin, cells will be incubated for 2 hours with 1/ a chicken polyclonal antibody anti microtubule-associated-protein 2 (MAP-2, RRID:AB_2138153), diluted at 1/1000 in PBS containing 1% fetal calf serum and 0.1% of saponin (this antibody allows the specific staining of neuronal cell bodies and neurites; and allow the study of neuronal cell death and neurite network) or 2/ a mouse monoclonal antibody anti-phospho Tau (AT100, RRID:AB_223652) at dilution of 1/400 in PBS containing 1% fetal calf serum and 0.1% of saponin, hours after intoxication, the hippocampal neurons were fixed by a cold solution of ethanol (95%) and acetic acid (5%) for 5 min at -20°C
, Synapses evaluation 24 hours after intoxication, the hippocampal neurons were fixed by a cold solution of ethanol (95%) and acetic acid (5%) for 5 min at -20°C. After permeabilization with 0.1% of saponin, cells were incubated for 2 hours with: 1/ a mouse monoclonal antibody anti post synaptic density 95kDa (PSD95, RRID:AB_300453) at a dilution of 1/100 in PBS containing 1% fetal calf serum and 0.1% of saponin or 2/ a rabbit polyclonal antibody anti-synaptophysin (SYN, RRID:AB_306124) at the dilution of 1/100 in PBS containing 1% fetal calf serum and 0.1% of saponin. These antibodies were revealed with Alexa Fluor 488 goat anti mouse IgG and Alexa Fluor 568 goat anti-rabbit IgG at the dilution 1/400 in PBS containing 1% FCS, 0.1% saponin, the dilution 1/400 in PBS containing 1% FCS, 0.1% saponin, for 1 hour at room temperature
, Synapses evaluation were performed automatically by using Custom module editor (Molecular Devices). The total number of synapses (overlapping between PSD95/SYN) was then assessed
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