Immunofluorescence Analysis of Hippocampal Synaptic Proteins

Immunofluorescence experiments were performed according to [73 (link)]. Briefly, hippocampal primary cultures at DIV14 (see above) were fixed in 4% paraformaldehyde (PFA) and 4% sucrose solution for 30 min, followed by permeabilization with phosphate-buffered saline (PBS) at pH 7.4, containing 0.5% Triton X-100, for 3 min. Co-cultures were first blocked for 1 h in PBS containing 1% BSA, 0.2% Triton X-100, and subsequently incubated overnight at 4 °C with primary antibodies in PBS containing 1% BSA and 0.2% Triton X-100. The following antibodies were used: anti-GFP (AbCam Cambridge, UK Ab290), anti-PSD95 (AbCam Cambridge, UK Ab12093), anti-JIP1 (AbCam Cambridge, UK Ab24449), anti-β-arrestin2 (AbCam Cambridge, UK Ab31294), anti-JNK3 (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA #PA5-14421). Cells were finally incubated with secondary antibodies (AlexFluor Antibody, Thermo Fisher Scientific, Waltham, MA, USA) for 1 h at room temperature. A concentration of 2 mg/mL Hoechst (Thermo Fisher Scientific, Waltham, MA, USA, 33342) was used to stain nuclei. ProLong Glass Antifade Mountant (Thermo Fisher Scientific, Waltham, MA, USA) was used as a mounting agent.