Figure 4

In situ hybridization combined with immunohistochemistry on free floating sections using 400 ng of digoxigenin (DIG)-labeled mouse GPR125 antisense probe stained with Fast Red enzyme substrate (A-C), white cell nucleus staining with (1:1000) DAPI (A), green staining with the (1:400) epithelial-cell-specific pan-cytokeratin antibody (A and B) and dark blue (1:500) glial fibrillary acidic protein (GFAP) staining (B and C). A) The z-stack function of the confocal microscope rendered 9 layers together to form this 3-dimensional image illustrating GPR125 expression in the cytoplasm around the DAPI-stained nuclei. Separate images are provided for the 3 channels followed by the merged images in the final panel. B) At higher magnification (63× objective), staining for the cytokeratin protein follows the edge of GPR125-labeled cells which illustrates co-localization. C) To show that GPR125 staining is predominantly in the choroid plexus, GFAP was used to stain ependymal cells which line the walls of the ventricle.