Expression pattern of KCa1.1 channels in MLIs. (A) Confocal immunofluorescence image of a sagittal cerebellar slice from a nNOS-ChR2 BAC mouse stained with an antibody targeting Slo1 KCa1.1 channels. The signal is present at the PC soma as well as throughout the molecular layer. (B–F) Maximum intensity projection images (37 planes at 0.4 um interval) from dual GFP/GCaMP3 and Slo1 staining following the ckit:cre sparse GCaMP3 expression strategy. The green channel displays the GFP/GCaMP3 signals while the red channel displays the Slo1 signals. (B) Superposition of the two channels. (C) Slo1 signal extracted by applying a digital mask from the GFP channel to the Slo1 channel. (D) Zoom of the axon initial segment, indicated by white arrows. (E) Thresholding applied to the Slo1 signal suggests channel clustering, as indicated by white arrows in F. Calibration bars were 20 μm in A, 10 μm in B, C, and E, and 5 μm in D and F.