Alexa fluor 488 anti guinea pig igg

Cortical cultures were fixed in supercold methanol (−20 °C), washed with PBS and blocked with 5% BSA, 5% NGS in PBS for 30 min. They were incubated with primary antibodies against MAP2 (1:2000, #188004; Synaptic Systems, Germany), GFAP (1:1000, #3670; Cell Signalling Technology), tyrosine hydroxylase (1:500, ab112; abcam) and GluN1 (1:500, ab9864; Merck Millipore), overnight at 4 °C. Sections were stained with secondary antibodies Alexa Fluor 488 anti-rabbit IgG, Alexa Fluor 488 anti-mouse IgG or Alexa Fluor 488 anti-guinea pig IgG (all Thermo Fisher Scientific, diluted 1:500) for 2 h at room temperature under minimal light conditions, washed with PBS and mounted in DAPI mounting media. Five images per coverslip were taken on a confocal microscope (SP2-AOBS, Leica). Analysis of the slides was performed with the experimenter blind to the experimental group. Dendrite lengths were measured using ImageJ. For receptors images were taken at x64 (with oil) on a fluorescence microscope (Leica SP5II) after excitation at 488 nm. Using ImageJ, images were converted to RGB files then measurements were taken of the mean grey value of each image providing an average of the relative intensity of the staining. Measurements were verified by cell counts based on DAPI staining. Background levels of fluorescence were ascertained in the absence of cells, primary antibody and/or secondary antibody.