Alexa fluor 488 anti sheep

To examine surface AMPA receptor labelling, staining was performed on rat hippocampal cultures (10-12 DIV) treated with vehicle or poly I:C (25μg /ml) for 1h, then immediately fixed with ice cold 4% paraformaldehyde for 10 minutes followed by blocking non-specific binding using a blocking buffer solution (5% FBS v/v and 1% BSA w/v in PBS, all Sigma, UK) for 30 minutes. The cultures were then incubated with a N-terminal GluA1antibody (1:100 in blocking buffer, kindly donated by Dr A. Irving, see Moult et al., 2010 (link)) at 4°C overnight followed by corresponding secondary antibodies (Alexa Fluor 488 anti-sheep, 1:200 dilution, Thermo Fisher, UK) in blocking solution for 60 min at room temperature. Image acquisition was performed taking random images with an Olympus BX51W1 microscope with a Q-imaging digital camera, with images acquired using WinFluor v3.4.4 imaging software (J. Dempster, University of Strathclyde, Glasgow, UK). GluA1 fluorescence intensity from vehicle and poly I:C treated cultures was measured using ImageJ software (NIH, USA) with 50 μm regions of interest added to 5 randomly selected neurites per coverslip. For neurites, n represents the number of neurites from at least three separate cultures, with all data expressed as mean ± S.E.M.