Distribution of glutamate receptors in Purkinje neurons of the cerebellum from WT and AP-4 ε KO mice.

<p>(A-C) Sections of the cerebellum from WT and AP-4 ε KO mice were co-immunostained with antibodies to calbindin (red) and δ2R (AB_2571601 from Frontier Institute Co., ltd) (A), GluA2 (SAB4501295 from Sigma) (B), or GluA1 (AB1504 from Millipore) (C). Staining for δ2R was done on sections treated with pepsin and staining for GluA1 on sections treated at 85°C with sodium-citrate buffer, in both cases for antigen retrieval. Similar results were obtained using samples that were not treated with high temperature sodium-citrate buffer (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007363#pgen.1007363.s005" target="_blank">S5C and S5D Fig</a>) and using a different antibody to GluA1 (antibody ab31232 from Abcam) (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007363#pgen.1007363.s005" target="_blank">S5A and S5B Fig</a>). Mo: molecular layer; Gr: granular layer, Pc: Purkinje cell layer, DCN: deep cerebellar nucleus. DAPI (blue) was used to stain the nuclei. Bars: 50 μm. Examples of spheroids are indicated by arrowheads. Single-channel images are shown in inverted grayscale. Notice the presence of δ2R and GluA2, and the absence of GluA1, in calbindin-positive spheroids in the DCN of AP-4 ε KO.