Figure 1.

Ser293-P antibody specifically recognizes Cx36 in the mouse retinal wholemounts. Confocal images show the lamina of the inner plexiform layer (IPL) adjacent to the retinal ganglion cell bodies. (A) mCx36 (blue) staining in wt retina (left). Magnified region shows strong punctate labeling (middle). No punctate labeling was present in Cx36KO retina, though there was some non-specific labeling of blood vessels by secondary anti-mouse antibodies (arrows). Their stereotypic localization of vascular elements in the IPL was used as a marker for specific retinal strata. The brightness of mCx36 puncta was increased for presentation purposes only to reveal all Cx36-positive plaques. (B) In the same region of wt retina as in (A), Ser293-P staining was present in wt retina, with similar punctate labeling. No Ser293-P staining was present in Cx36KO retina. The degree of phosphorylation, reflected by the intensity of the Ser293-P labeling, varied across Cx36 plaques. (C) The magnified region (middle) shows that the vast majority of Ser293-P-positive puncta colocalized with mCx36-postive puncta in wt retina. 97.8 ± 0.3% were colocalized with mCx36 (3 retinas × 3 samples = 976 detectably phosphorylated plaques). Scale bars: 10 μm, left and right columns; 2 μm, middle column.