Alexa 568 conjugated anti mouse secondary antibody

Immunocytochemistry was performed on 7–12 day old cultured (DIC) hippocampal neurons. Before labelling, neurons were washed with HEPES buffered saline containing glycine (0.01 mM) and treated with G1 for 30 min at room temperature (21–23 °C). For antagonist studies, neurons were pre-treated with pharmacological inhibitors for 30 min before G1 application. To label surface GluA1, neurons were incubated with an antibody against the N-terminal region of GluA1 (sheep anti-GluA1; 1:100; [47 (link)] at 4 °C, then fixed with 4% paraformaldehyde for 5 min. Surface GluA1 immunostaining was visualized by addition of an appropriate fluorescently conjugated anti-sheep secondary antibody (1:250, Life Technologies, UK) for 30 min. In a subset of experiments, neurons were permeabilized with 0.1% Triton X-100 (5 min) after GluA1 immunolabelling and fixed with 4% paraformaldehyde. A second primary antibody was applied to compare GluA1 surface immunostaining relative to PSD-95 (mouse anti-PSD-95; 1:500; Thermo Fisher, UK). An Alexa 568-conjugated anti-mouse secondary antibody (1:200; Thermo Fisher, UK) was then used to visualize PSD-95 labelling [46 (link)]. No labelling was observed after incubation with secondary antibodies alone.

HEK293T cells were cultured on confocal slides, transfected with plasmids to express wild-type and Derlin-1 mutants. 36 hours later, cells were fixed with 2% paraformaldehyde at room temperature for 10 minutes, permeabilized with 0.1% Triton X-100 in PBS, washed three times in 0.5% bovine serum albumin and 0.15% glycine at (pH 7.4) in phosphate-buffered saline, reacted with anti-His antibody (ABclonal), and incubated with Alexa 568 conjugated anti-mouse secondary antibody (ThermoFisher). After washing with 0.5% bovine serum albumin and 0.15% glycine at (pH 7.4) in phosphate-buffered saline, the coverslips were mounted for confocal microscopy.