Retinal immunohistochemistry for optogenetics

At the end of the terminal experiment, mice were euthanized and retinas extracted for subsequent immunohistochemistry to confirm retinal expression patterns of the optogenes. Immunohistochemistry of cryosections were similar to that described previously^{26 (link)}. In brief, retinas or eyecups were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 30 min. Antibodies were diluted in a blocking solution containing 1% Triton-X and 2% donkey serum. Sections were incubated overnight at 4 °C in primary antibody and 2 h in secondary antibody at room temperature. The following primary antibodies were used: rabbit anti-tRFP (1:1000; Evrogen; AB234), rabbit anti-melanopsin (1:1000; Advanced Targeting Systems; AB-N39), goat anti-ChAT (1:100; Millipore; AB144P) and mouse anti-Goα (1:1000; Millipore; mab3073). Secondary antibodies were always from donkey and either conjugated to Alexa 488 or Alexa 594 (1:400; Invitrogen). Alexa 488 conjugated to streptavidin was used to visualize cells injected with biocytin during patch-clamp experiments (1:400; Invitrogen; S-11223). Nuclei were stained with 10 μg/ml DAPI (Roche). Micrographs were taken on a Zeiss LSM 880. Processing of image stacks was done using ImageJ (Rasband WS, United States National Institutes of Health, Bethesda, Maryland, US).