Fig. 1.

Quantification methods. A: a representative confocal image of transient receptor potential melastatin 1 (TRPM1) staining shows the regions of interest used to find the average intensities for the different layers. For this and the following figures: ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. The region of interest in the INL includes only the top tiers where ON bipolar cells reside, and the region of interest in the IPL is divided into IPL-OFF (which also includes the lower tiers of the INL) and IPL-ON. B and C: a representative confocal image of TRPM1 staining shows the region of interest for counting puncta in the OPL (B), and the puncta selected by Volocity (C; magenta rectangles). D: a dissociated ON bipolar cell immunostained for Na⁺-K⁺-ATPase (cyan; outlines the plasma membrane) and for TRPM1 (red). The cell was also stained with 4,6-diamidino-2-phenylindole (DAPI; blue) to locate the nucleus. Five lines perpendicular to the plasma membrane were drawn, and the intensity profiles for two of them are shown in E. E: intensity profiles (along lines 8 and 9 shown in D) plot the intensity of each pixel proceeding from the extracellular to the intracellular space. The profiles are aligned by the peak of the Na⁺-K⁺-ATPase stain (vertical line). The dotted boxed region in each profile overlaps with the nucleus and, therefore, shows the background level of TRPM1 staining in the cell.