Fig. 3.

Expression of Orai/Stim in optic nerve astrocytes. Double immunofluorescence labelling for Orai1 (a, green), Stim1 (b, green) and Stim2 (c, green), with GFAP to identify astrocytes (a–c, red), in WT optic nerve sections (ai–iv, bi–iv, ci–iv) and explant cultures (av, bv, cv). Confocal micrographs illustrate single channels (ai, aii, bi, bii, ci, cii) and merged cannels (aiii, biii, ciii). Expression of Orai1 and Stim1 is localized to astrocyte processes, whereas astrocytes were immunonegative for Stim2, as demonstrated by the colocalization channels, illustrating voxels in which green and red channels are of equal intensity and appear yellow (aiv, biv, civ). Astrocytes in explant cultures are immunopositive for Orai1 (av) and Stim1 (bv), but are immunonegative for Stim2 (cv). No immunostaining was observed in negative controls that were pre-incubated in blocking peptides for Orai1 (inset, aiii), Stim1 (inset, biii) and Stim2 (inset, ciii). Nuclei are stained with Hoechst blue. Scale bars a, b 10 µm, c 20 μm