Sucrose 4 formaldehyde

At DIV19–21, neurons used for Arc induction and degradation experiments were washed twice with 37°C 4% sucrose/1× phosphate-buffered-saline (PBS; 10×: 1.4 M NaCl, 26.8 mM KCl, 62 mM Na₂HPO₄, 35.3 mM KH₂PO₄, pH 7.4), then fixed for 15 min with 4% sucrose/4% formaldehyde (Thermo Fisher Scientific) in 1× PBS. Neurons were washed 3 × 5 min with 1× PBS, permeabilized for 10 min with 0.2% Triton X-100 (Amresco, Solon, OH, USA) in 1× PBS, and blocked for 30 min in 5% normal donkey serum (Jackson ImmunoResearch) in 1× PBS. Neurons were then incubated in primary antibody diluted in block for 1 h at RT, washed 3 × 5 min in 1× PBS, and incubated in secondary antibody diluted in block for 1 h at RT. Neurons on coverslips were mounted on glass slides in Fluoromount (Thermo Fisher Scientific) and dried overnight at RT. For live-labeling of surface GluA1 receptors (Shepherd et al., 2006 (link)), neurons were washed twice with 10°C 4% sucrose/1× PBS, then incubated in anti-GluA1-NT diluted in MEM containing 2% GlutaMAX, 2% B-27, 15 mM HEPES (Thermo Fisher Scientific), 1 mM sodium pyruvate (Thermo Fisher Scientific), and 33 mM glucose at 10°C for 20 min. Neurons were then fixed and incubated in Alexa Fluor 555 before permeabilization to label only surface GluA1. Following this, neurons were permeabilized and further immunostained as above.