Cryosections were prepared from adult enucleated eyes or whole larvae and immunostained as previously described (10 (link),52 (link)). Primary antibodies used were 4D2 (1:200; Abcam, Cambridge, UK; ab98887), anti-UV opsin, anti-blue opsin, anti-red opsin (1:200; gifted by Professor David Hyde, University of Notre Dame), anti-usherin-C (1:500; Novus Biological, Littleton, CO, USA; 27 640 002), anti-usherin-N (1:200; gifted by Dr Jennifer Phillips, University of Oregon), anti-LC3 (1:200; Thermo Fisher Scientific, Waltham, MA, USA; PA1-16930), anti-centrin (1:500; Merck-Millipore; 04-1624) or anti-acetylated tubulin (1:200; Sigma-Aldrich, St Louis, MO, USA; T7451). Primary antibodies and appropriate secondary Alexa Fluor antibodies (1:500; Thermo Fisher Scientific) were diluted in antibody solution. The slides were imaged using a Leica LSM 700 confocal microscope or a Zeiss Axio Imager fluorescence microscope equipped with an Axiocam MRC5 camera. Rhodopsin levels were quantified using ImageJ. For this, all pictures were taken using the same settings after which the region of interest (adult retina: rod outer segments, larval retina: inner segment and ONL) was defined manually and assessed using the mean pixel intensity measurement.
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