Primary neuronal cultures were fixed with 4% PFA/4% sucrose solution for 10 min at RT, washed with PBS, and permeabilized with 0.2% TX‐100 in PBS for 10 min. Then, cells were incubated for 1 h in blocking solution (2% glycine, 0.2% gelatine, 2% BSA, and 50 mM NH4Cl (pH 7.4)) and primary antibodies were applied overnight at 4°C. Next, the coverslips were incubated with secondary antibodies diluted 1:500. Coverslips were mounted with Mowiol 4‐88 (Merck Chemicals). For detection of Jacob‐CREB and Jacob‐LMO4 co‐localization in STED imaging, a heat‐based antigen retrieval protocol was used.
For assessing surface expression of AMPA receptors, dissociated hippocampal neurons were incubated for 10 min at RT with anti‐GluA1 antibody diluted in Tyrode's buffer (128 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 4.2 mM NaHCo3, 15 mM HEPES, 20 mM glucose, pH 7.2–7.4), rinsed, and fixed for 10 min at RT with 4% PFA‐sucrose, and subsequently stained with other antibodies as described above.
For assessing surface expression of AMPA receptors, dissociated hippocampal neurons were incubated for 10 min at RT with anti‐GluA1 antibody diluted in Tyrode's buffer (128 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 4.2 mM NaHCo3, 15 mM HEPES, 20 mM glucose, pH 7.2–7.4), rinsed, and fixed for 10 min at RT with 4% PFA‐sucrose, and subsequently stained with other antibodies as described above.
