Fig. 2.

Colocalization of KAR subunits and group I mGlu subunits in rat cortical cultures. Immunocytochemistry was performed on rat cortical cultures. Images were taken with a 100×/1.3 oil objective lens using a laser confocal microscope. Positive fluorescent staining was observed for mGlu1 (A and D), mGlu5 (B, C, and E), GluK4 (A and B), and GluK5 (D and E). The high magnification overlapping confocal images (A, B, D, and E) more clearly show the colocalization of the GluK and mGlu subunits. (C) overlap of synapsin and mGlu5 immunostains in the dendrites of the cortical neurons. F, Quantification of the colocalization of the KAR subunits and the group I mGlu subunits in the cortical cultures (n ≥ 3). Positive designates a positive control in which GluK5 and synapsin are stained with 594 and 488 conjugated secondary antibodies, respectively. Negative designates a negative control in which the primary antibodies are omitted. Only background staining was seen in such cultures exposed to the secondary antibodies only (data not shown) (Alexa Fluor 488 and Alexa Fluor 594). The dashed line at 0.3 represents the Mander’s coefficient threshold cutoff for background labeling as defined by the negative control. All the colocalization pairs showed a significantly higher Mander’s coefficient, compared with the negative control (**P < 0.01; one-way ANOVA with selected Dunnett’s post-tests). The bar = 20 μm.