Alexa fluor 488 goat anti mouse ab

Mouse cortical cells were seeded at 75,000 cells/well on poly-L-lysine (Sigma-Aldrich) coated coverslips. Cells were fixed with 4% paraformaldehyde (PFA) for 10 min and washed three times with PBS (10 min each) to remove PFA traces. Cells were permeabilized with 0.1% Triton X-100 and washed thrice with PBS. Coverslips were incubated with blocking solution (5% fetal bovine serum, 1% BSA and 0.02% sodium azide) over night at 4 °C. Subsequently, cells were incubated for 2 h at room temperature (RT) in a hydration chamber with 1:10 mouse anti-GluN2B Ab (Neuromab), 1/700 rabbit anti-GluN2B Ab (Abcam), 1/500 mouse anti-PSD95 Ab (Cell signalling), 1/500 rabbit anti-HRI Ab (Abcam) or 1/500 mouse anti-GluN1 Ab (Synaptic system). After primary Ab incubation, cells were incubated with 1:1000 Alexa Fluor 488 goat anti-mouse Ab or 1:1000 Alexa Fluor 565 goat anti-rabbit Ab (Invitrogen) for 1 h at RT and washed three times (5 min each) with PBS. Digital images were taken with a Leica TCS SP confocal microscope and analysed with Leica confocal software (Heidelberg, Germany). Co-localization analyses were performed with Image J software, adjusting the threshold of 10 dendrites from 6 different neurons for each experimental condition from 3 independent experiments.